Methods for producing secreted ligand-binding fusion proteins

ABSTRACT

Methods for producing secreted receptor analogs and biologically active peptide dimers are disclosed. The methods for producing secreted receptor analogs and biologically active peptide dimers utilize a DNA sequence encoding a receptor analog or a peptide requiring dimerization for biological activity joined to a dimerizing protein. The receptor analog includes a ligand-binding domain. Polypeptides comprising essentially the extracellular domain of a human PDGF receptor fused to dimerizing proteins, the portion being capable of binding human PDGF or an isoform thereof, are also disclosed. The polypeptides may be used within methods for determining the presence of and for purifying human PDGF or isoforms thereof.

CROSS REFERENCE TO RELATED APPLICATION

This is a continuation application and claims the benefit of U.S. patentapplication Ser. No. 09/435,059, filed Oct. 25, 1999, now pending; whichis a continuation of U.S. patent application Ser. No. 08/980,400, filedNov. 26, 1997, now U.S. Pat. No. 6,018,026; which is a divisional ofU.S. patent application Ser. No. 08/477,329, file Jun. 7, 1995, now U.S.Pat. No. 5,750,375; which is a continuation of U.S. patent applicationSer. No. 08/180,195, filed Jan. 11, 1994, now U.S. Pat. No. 5,567,584;which is a continuation of U.S. patent application Ser. No. 07/634,510,filed Dec. 27, 1990, now abandoned; which is a continuation-in-part ofU.S. patent application Ser. No. 07/347,291, filed May 2, 1989, now U.S.Pat. No. 5,155,027; which is a continuation-in-part of U.S. patentapplication Ser. No. 07/146,877, filed Jan. 22, 1988, now abandoned, thedisclosures of which are incorporated herein by reference. Thisapplication claims the benefit under 35 U.S.C. §119(a) of EuropeanPatent Application No. 89100787.4, filed Jan. 18, 1989.

TECHNICAL FIELD

The present invention is generally directed toward the expression ofproteins, and more specifically, toward the expression of growth factorreceptor analogs and biologically active dimerized polypeptide fusions.

BACKGROUND OF THE INVENTION

In higher eucaryotic cells, the interaction between receptors andligands (e.g., hormones) is of central importance in the transmission ofand response to a variety of extracellular signals. It is generallyaccepted that hormones and growth factors elicit their biologicalfunctions by binding to specific recognition sites (receptors) in theplasma membranes of their target cells. Upon ligand binding, a receptorundergoes a conformational change, triggering secondary cellularresponses that result in the activation or inhibition of intracellularprocesses. The stimulation or blockade of such an interaction bypharmacological means has important therapeutic implications for a widevariety of illnesses.

Ligands fall into two classes: those that have stimulatory activity,termed agonists; and those that block the effects elicited by theoriginal ligands, termed antagonists. The discovery of agonists thatdiffer in structure and composition from the original ligand may bemedically useful. In particular, agonists that are smaller than theoriginal ligand may be especially useful. The bioavailability of thesesmaller agonists may be greater than that of the original ligand. Thismay be of particular importance for topical applications and forinstances when diffusion of the agonist to its target sites is inhibitedby poor circulation. Agonists may also have slightly different spectraof biological activity and/or different potencies, allowing them to beused in very specific situations. Agonists that are smaller andchemically simpler than the native ligand may be produced in greaterquantity and at lower cost. The identification of antagonists whichspecifically block, for example, growth factor receptors has importantpharmaceutical applications. Antagonists that block receptors againstthe action of endogenous, native ligand may be used as therapeuticagents for conditions including atherosclerosis, autocrine tumors,fibroplasia and keloid formation.

The discovery of new ligands that may be used in pharmaceuticalapplications has centered around designing compounds by chemicalmodification, complete synthesis, and screening potential ligands bycomplex and costly screening procedures. The process of designing a newligand usually begins with the alteration of the structure of theoriginal effector molecule. If the original effector molecule is knownto be chemically simple, for example, a catecholamine or prostaglandin,the task is relatively straightforward. However, if the ligand isstructurally complex, for example, a peptide hormone or a growth factor,finding a molecule which is functionally equivalent to the originalligand becomes extremely difficult.

Currently, potential ligands are screened using radioligand bindingmethods (Lefkowitz et al., Biochem. Biophys. Res. Comm. 60: 703-709,1974; Aurbach et al., Science 186: 1223-1225, 1974; Atlas et al., Proc.Natl. Acad. Sci. USA 71: 4246-4248, 1974). Potential ligands can bedirectly assayed by binding the radiolabeled compounds to responsivecells, to the membrane fractions of disrupted cells, or to solubilizedreceptors. Alternatively, potential ligands may be screened by theirability to compete with a known labeled ligand for cell surfacereceptors.

The success of these procedures depends on the availability ofreproducibly high quality preparations of membrane fractions or receptormolecules, as well as the isolation of responsive cell lines. Thepreparation of membrane fractions and soluble receptor moleculesinvolves extensive manipulations and complex purification steps. Theisolation of membrane fractions requires gentle manipulation of thepreparation, a procedure which does not lend itself to commercialproduction. It is very difficult to maintain high biological activityand biochemical purity of receptors when they are purified by classicalprotein chemistry methods. Receptors, being integral membrane proteins,require cumbersome purification procedures, which include the use ofdetergents and other solvents that interfere with their biologicalactivity. The use of these membrane preparations in ligand bindingassays typically results in low reproducibility due to the variabilityof the membrane preparations.

As noted above, ligand binding assays require the isolation ofresponsive cell lines. Often, only a limited subset of cells isresponsive to a particular agent, and such cells may be responsive onlyunder certain conditions. In addition, these cells may be difficult togrow in culture or may possess a low number of receptors. Currentlyavailable cell types responsive to platelet-derived growth factor(PDGF), for example, contain only a low number (up to 4×10⁵; seeBowen-Pope and Ross, J. Biol. Chem. 257: 5161-5171, 1982) of receptorsper cell, thus requiring large numbers of cells to assay PDGF analogs orantagonists.

Presently, only a few naturally-occurring secreted receptors, forexample, the interleukin-2 receptor (IL-2-R) have been identified. Rubinet al. (J. Immun. 135: 3172-3177, 1985) have reported the release oflarge quantities of IL-2-R into the culture medium of activated T-celllines. Bailon et al. (Bio/Technology 5: 1195-1198, 1987) have reportedthe use of a matrix-bound interleukin-2 receptor to purify recombinantinterleukin-2.

Three other receptors have been secreted from mammalian cells. Theinsulin receptor (Ellis et al., J. Cell Biol. 150: 14a, 1987), the HIV-1envelope glycoprotein cellular receptor CD4 (Smith et al., Science 238:1704-1707, 1987), the murine IL-7 receptor (Cell 60: 941-951, 1990) andthe epidermal growth factor (EGF) receptor (Livneh et al., J. Biol.Chem. 261: 12490-12497, 1986) have been secreted from mammalian cellsusing truncated cDNAs that encode portions of the extracellular domains.

Naturally-occurring, secreted receptors have not been widely identified,and there have been only a limited number of reports of secretedrecombinant receptors. Secreted receptors may be used in a variety ofassays, which include assays to determine the presence of ligand inbiological fluids and assays to screen for potential agonists andantagonists. Current methods for ligand screening and ligand/receptorbinding assays have been limited to those using preparations of wholecells or cell membranes for as a source for receptor molecules. The lowreproducibility and high cost of producing such preparations does notlend itself to commercial production. There is therefore a need in theart for a method of producing secreted receptors. There is a furtherneed in the art for an assay system that permits high volume screeningof compounds that may act on higher eucaryotic cells via specificsurface receptors. This assay system should be rapid, inexpensive andadaptable to high volume screening. The present invention discloses sucha method and assay system, and further provides other relatedadvantages.

DISCLOSURE OF INVENTION

Briefly stated, the present invention discloses methods for producingsecreted receptor analogs, including receptor analogs and secretedplatelet-derived growth factor receptor (PDGF-R) analogs. In addition,the present invention discloses methods for producing secretedbiologically active dimerized polypeptide fusions.

Within one aspect of the invention a method for producing a secretedPDGF-R analog is disclosed, comprising (a) introducing into a eukaryotichost cell a DNA construct comprising a transcriptional promoteroperatively linked to a secretory signal sequence followed downstream ofand in proper reading frame with a DNA sequence encoding at least aportion of the ligand-binding domain of a PDGF-R, the portion includinga ligand-binding domain; (b) growing the host cell in an appropriategrowth medium under physiological conditions to allow the secretion of aPDGF-R analog encoded by said DNA sequence; and (c) isolating the PDGF-Ranalog from the host cell.

Within one embodiment of the present invention, a PDGF-R analogcomprising the amino acid sequence of FIG. 1 (Sequence ID Numbers 1 and2) from isoleucine, number 29, to methionine, number 441, is secreted.Within another embodiment, a PDGF-R analog comprising the amino acidsequence of FIG. 1 (Sequence ID Numbers 1 and 2) from isoleucine, number29 to lysine, number 531 is secreted. Within yet another embodiment ofthe invention, a PDGF-R analog comprising the amino acid sequence ofFIG. 11 (Sequence ID Numbers 35 and 36) from glutamine, number 24 toglutamic acid, number 524 is secreted.

Yet another aspect of the present invention discloses a method forproducing a secreted, biologically active dimerized polypeptide fusion.The method generally comprises a) introducing into a eukaryotic hostcell a DNA construct comprising a transcriptional promoter operativelylinked to a secretory signal sequence followed downstream by and inproper reading frame with a DNA sequence encoding a non-immunoglobulinpolypeptide requiring dimerization for biological activity joined to adimerizing protein; (b) growing the host cell in an appropriate growthmedium under physiological conditions to allow the secretion of adimerized polypeptide fusion encoded by said DNA sequence; and (c)isolating the biologically active dimerized polypeptide fusion from thehost cell.

Within one embodiment, the dimerizing protein is yeast invertase. Withinanother embodiment, the dimerizing protein is at least a portion of animmunoglobulin light chain. Within another embodiment, the dimerizingprotein is at least a portion of an immunoglobulin heavy chain.

In another aspect of the invention, a method is disclosed for producinga secreted, biologically active dimerized polypeptide fusion, comprising(a) introducing into a eukaryotic host cell a first DNA constructcomprising a transcriptional promoter operatively linked to a firstsecretory signal sequence followed downstream by and in proper readingframe with a first DNA sequence encoding a non-immunoglobulinpolypeptide requiring dimerization for biological activity joined to animmunoglobulin light chain constant region; (b) introducing into thehost cell a second DNA construct comprising a transcriptional promoteroperatively linked to a second secretory signal sequence followeddownstream by and in proper reading frame with a second DNA sequenceencoding an immunoglobulin heavy chain constant region domain selectedfrom the group consisting of C_(H)1, C_(H)2, C_(H)3, and C_(H)4; (c)growing the host cell in an appropriate growth medium underphysiological conditions to allow the secretion of a dimerizedpolypeptide fusion encoded by said first and second DNA sequences; and(d) isolating the dimerized polypeptide fusion from the host cell. Inone embodiment, the second DNA sequence further encodes animmunoglobulin heavy chain hinge region wherein the hinge region isjoined to the heavy chain constant region domain. In a preferredembodiment, the second DNA sequence further encodes an immunoglobulinvariable region joined upstream of and in proper reading frame with theimmunoglobulin heavy chain constant region.

In another aspect of the invention, a method is disclosed for producinga secreted, biologically active dimerized polypeptide fusion, comprising(a) introducing into a eukaryotic host cell a first DNA constructcomprising a transcriptional promoter operatively linked to a firstsecretory signal sequence followed downstream by and in proper readingframe with a first DNA sequence encoding a non-immunoglobulinpolypeptide requiring dimerization for biological activity joined to animmunoglobulin heavy chain constant region domain selected from thegroup consisting of C_(H)1, C_(H)2, C_(H)3, and C_(H)4; (b) introducinginto the host cell a second DNA construct comprising a transcriptionalpromoter operatively linked to a second secretory signal sequencefollowed downstream by and in proper reading frame with a second DNAsequence encoding an immunoglobulin light chain constant region; (c)growing the host cell in an appropriate growth medium underphysiological conditions to allow the secretion of a dimerizedpolypeptide fusion encoded by said first and second DNA sequences; and(d) isolating the dimerized polypeptide fusion from the host cell. Inone embodiment, the first DNA sequence further encodes an immunoglobulinheavy chain hinge region wherein the hinge region is joined to the heavychain constant region domain. In a preferred embodiment, the second DNAsequence further encodes an immunoglobulin variable region joinedupstream of and in proper reading frame with the immunoglobulin lightchain constant region.

In another aspect of the invention, a method is disclosed for producinga secreted, biologically active dimerized polypeptide fusion, comprising(a) introducing into a eukaryotic host cell a DNA construct comprising atranscriptional promoter operatively linked to a secretory signalsequence followed downstream by and in proper reading frame with a DNAsequence encoding a non-immunoglobulin polypeptide requiringdimerization for biological activity joined to an immunoglobulin heavychain constant region domain selected from the group consisting ofC_(H)1, C_(H)2, C_(H)3, and C_(H)4; (b) growing the host cell in anappropriate growth medium under physiological conditions to allow thesecretion of a dimerized polypeptide fusion encoded by said first andsecond DNA sequences; and (c) isolating the biologically activedimerized polypeptide fusion from the host cell. In one embodiment, theDNA sequence further encodes an immunoglobulin heavy chain hinge regionwherein the hinge region is joined to the heavy chain constant regiondomain.

In another aspect of the invention, a method is disclosed for producinga secreted, biologically active dimerized polypeptide fusion, comprising(a) introducing into a eukaryotic host cell a first DNA constructcomprising a transcriptional promoter operatively linked to a firstsecretory signal sequence followed downstream by and in proper readingframe with a first DNA sequence encoding a first polypeptide chain of anon-immunoglobulin polypeptide dimer requiring dimerization forbiological activity joined to an immunoglobulin heavy chain constantregion domain, selected from the group consisting of C_(H)1, C_(H)2,C_(H) ³, and C_(H)4; (b) introducing into the host cell a second DNAconstruct comprising a transcriptional promoter operatively linked to asecond secretory signal sequence followed downstream by and in properreading frame with a second DNA sequence encoding a second polypeptidechain of the non-immunoglobulin polypeptide dimer joined to animmunoglobulin light chain constant region domain; (c) growing the hostcell in an appropriate growth medium under physiological conditions toallow the secretion of a dimerized polypeptide fusion encoded by saidfirst and second DNA sequences wherein said dimerized polypeptide fusionexhibits biological activity characteristic of said non-immunoglobulinpolypeptide dimer; and (d) isolating the dimerized polypeptide fusionfrom the host cell. In one embodiment the first DNA sequence furtherencodes an immunoglobulin heavy chain hinge region domain wherein thehinge region is joined to the immunoglobulin heavy chain constant regiondomain.

Within one embodiment of the present invention, a biologically activedimerized polypeptide fusion comprising the amino acid sequence of FIG.1 (Sequence ID Numbers 1 and 2) from isoleucine, number 29, tomethionine, number 441, is secreted. Within another embodiment, abiologically active dimerized polypeptide fusion comprising the aminoacid sequence of FIG. 1 (Sequence ID Numbers 1 and 2) from isoleucine,number 29 to lysine, number 531 is secreted. Within another embodimentof the invention, a biologically active dimerized polypeptide fusioncomprising the amino acid sequence of FIG. 11 (Sequence ID Numbers 35and 36) from glutamine, number 24 to glutamic acid, number 524 issecreted. Within yet another embodiment of the invention, a biologicallyactive dimerized polypeptide fusion comprising the amino acid sequenceof FIG. 1 (Sequence ID Numbers 1 and 2) from isoleucine, number 29 tolysine, number 531 dimerized to the amino acid sequence of FIG. 11(Sequence ID Numbers 35 and 36) from glutamine, number 24 to glutamicacid, number 524 is secreted.

In yet another aspect of the invention, a method is disclosed forproducing a secreted receptor analog, comprising (a) introducing into aeukaryotic host cell a DNA construct comprising a transcriptionalpromoter operatively linked to at least one secretory signal sequencefollowed downstream by and in proper reading frame with a DNA sequenceencoding a ligand-binding domain of a receptor requiring dimerizationfor biological activity joined to a dimerizing protein; (b) growing thehost cell in an appropriate growth medium under physiological conditionsto allow the secretion of a receptor analog encoded by said DNAsequence; and (c) isolating the receptor analog from the host cell.

In yet another aspect of the invention, a method is disclosed forproducing a secreted receptor analog, comprising (a) introducing into aeukaryotic host cell a first DNA construct comprising a transcriptionalpromoter operatively linked to a first secretory signal sequencefollowed downstream by and in proper reading frame with a first DNAsequence encoding a ligand-binding domain of a receptor requiringdimerization for biological activity joined to an immunoglobulin lightchain constant region; (b) introducing into the host cell a second DNAconstruct comprising a transcriptional promoter operatively linked to asecond secretory signal sequence followed downstream by and in properreading frame with a second DNA sequence encoding an immunoglobulinheavy chain constant region domain, selected from the group consistingof C_(H)1, C_(H)2, C_(H)3, and C_(H)4; (c) growing the host cell in anappropriate growth medium under physiological conditions to allow thesecretion of a receptor analog encoded by said first and second DNAsequences; and (d) isolating the receptor analog from the host cell. Inone embodiment, the second DNA sequence further encodes animmunoglobulin heavy chain hinge region wherein the hinge region isjoined to the heavy chain constant region domain. In a preferredembodiment, the second DNA sequence further encodes an immunoglobulinvariable region joined upstream of and in proper reading frame with theimmunoglobulin heavy chain constant region.

In another aspect of the invention, a method is disclosed for producinga secreted receptor analog, comprising (a) introducing into a eukaryotichost cell a DNA construct comprising a transcriptional promoteroperatively linked to a secretory signal sequence followed downstream byand in proper reading frame with a DNA sequence encoding aligand-binding domain of a receptor requiring dimerization forbiological activity joined to an immunoglobulin heavy chain constantregion domain, selected from the group C_(H)1, C_(H)2, C_(H)3, andC_(H)4; (b) growing the host cell in an appropriate growth medium underphysiological conditions to allow the secretion of the receptor analog;and (c) isolating the receptor analog from the host cell. In oneembodiment, the DNA sequence further encodes an immunoglobulin heavychain hinge region wherein the hinge region is joined to the heavy chainconstant region domain.

In another aspect of the invention, a method is disclosed for producinga secreted receptor analog, comprising (a) introducing into a eukaryotichost cell a first DNA construct comprising a transcriptional promoteroperatively linked to a first secretory signal sequence followeddownstream of and in proper reading frame with a first DNA sequenceencoding a ligand-binding domain of a receptor requiring dimerizationfor biological activity joined to an immunoglobulin heavy chain constantregion domain, selected from the group C_(H)1, C_(H)2, C_(H)3, andC_(H)4; (b) introducing into the host cell a second DNA constructcomprising a transcriptional promoter operatively linked to a secondsecretory signal sequence followed downstream by and in proper readingframe with a second DNA sequence encoding an immunoglobulin light chainconstant region; (c) growing the host cell in an appropriate growthmedium under physiological conditions to allow the secretion of areceptor analog encoded by said first and second DNA sequences; and (d)isolating the receptor analog from the host cell. In one embodiment, thefirst DNA sequence further encodes an immunoglobulin heavy chain hingeregion wherein the hinge region is joined to the heavy chain constantregion domain. In a preferred embodiment, the second DNA sequencefurther encodes an immunoglobulin variable region joined upstream of andin proper reading frame with the immunoglobulin light chain constantregion.

In another aspect of the invention, a method is disclosed for producinga secreted receptor analog, comprising (a) introducing into a eukaryotichost cell a first DNA construct comprising a transcriptional promoteroperatively linked to a first secretory signal sequence followeddownstream in proper reading frame by a first DNA sequence encoding afirst polypeptide chain of a ligand-binding domain of a receptorrequiring dimerization for biological activity joined to animmunoglobulin heavy chain constant region domain, selected from thegroup C_(H)1, C_(H)2, C_(H)3, and C_(H)4; (b) introducing into the hostcell a second DNA construct comprising a transcriptional promoteroperatively linked to a second secretory signal sequence followeddownstream by and in proper reading frame with a second DNA sequenceencoding a second polypeptide chain of the ligand-binding domain of saidreceptor joined to an immunoglobulin light chain constant region domain;(c) growing the host cell in an appropriate growth medium underphysiological conditions to allow the secretion of a receptor analogencoded by said first and second DNA sequences; and (d) isolating thereceptor analog from the host cell. In one embodiment the first DNAsequence further encodes an immunoglobulin heavy chain hinge regiondomain wherein the hinge region is joined to the immunoglobulin heavychain constant region domain.

Host cells for use in the present invention include cultured mammaliancells and fungal cells. In a preferred embodiment strains of the yeastSaccharomyces cerevisiae are used as host cells. Within anotherpreferred embodiment cultured rodent myeloma cells are used as hostcells.

Within one embodiment of the present invention, a receptor analog is aPDGF-R analog comprising the amino acid sequence of FIG. 1 (Sequence IDNumbers 1 and 2) from isoleucine, number 29, to methionine, number 441.Within another embodiment a PDGF-R analog comprises the amino acidsequence of FIG. 1 (Sequence ID Numbers 1 and 2) from isoleucine, number29, to lysine, number 531. Within another embodiment of the invention, aPDGF-R analog comprises the amino acid sequence of FIG. 11 (Sequence IDNumbers 35 and 36) from glutamine, number 24 to glutamic acid, number524 is secreted. Within yet another embodiment of the invention, aPDGF-R analog comprises the amino acid sequence of FIG. 1 (Sequence IDNumbers 1 and 2) from isoleucine, number 29 to lysine, number 531 andthe amino acid sequence of FIG. 11 (Sequence ID Numbers 35 and 36) fromglutamine, number 24 to glutamic acid, number 524 is secreted.

PDGF-R analogs produced by the above-disclosed methods may be used, forinstance, within a method for determining the presence of human PDGF oran isoform thereof in a biological sample.

A method for determining the presence of human PDGF or an isoformthereof in a biological sample is disclosed and comprises (a) incubatinga polypeptide comprising a PDGF receptor analog fused to a dimerizingprotein with a biological sample suspected of containing PDGF or anisoform thereof under conditions that allow the formation ofreceptor/ligand complexes; and (b) detecting the presence ofreceptor/ligand complexes, and therefrom determining the presence ofPDGF or an isoform thereof. Suitable biological samples in this regardinclude blood, urine, plasma, serum, platelet and other cell lysates,platelet releasates, cell suspensions, cell-conditioned culture media,and chemically or physically separated portions thereof.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1G (Sequence ID Numbers 1 and 2) illustrate the nucleotidesequence of a representative PDGF β-receptor cDNA and the derived aminoacid sequence of the primary translation product and correspond toSequence ID Number 1). Numbers above the lines refer to the nucleotidesequence; numbers below the lines refer to the amino acid sequence.

FIG. 2 illustrates the construction of pBTL10, pBTL11 and pBTL12.

FIG. 3 illustrates the construction of pCBS22.

FIG. 4 illustrates the construction of pBTL13 and pBTL14.

FIG. 5 illustrates the construction of pBTL15

FIG. 6 illustrates the construction of pBTL22 and pBTL26.

FIG. 7 illustrates the construction of pSDL114. Symbols used are S.S.,signal sequence, C_(k), immunoglobulin light chain constant regionsequence; μ prom, μ promoter; μ enh, μ enhancer.

FIG. 8 illustrates the construction of pSDLB113. Symbols used are S.S.,signal sequence; C_(H)1, C_(H)2, C_(H)3, immunoglobulin heavy chainconstant region domain sequences; H, immunoglobulin heavy chain hingeregion sequence; M, immunoglobulin membrane anchor sequences; Cγ1M,immunoglobulin heavy chain constant region and membrane anchorsequences.

FIG. 9 illustrates the constructions pBTL115, pBTL114,pφ5V_(H)HuCγ1M-neo, plCφ5V_(κ)HuC_(κ)-neo. Symbols used are set forth inFIGS. 7 and 8, and also include L_(H), mouse immunoglobulin heavy chainsignal sequence; V_(H), mouse immunoglobulin heavy chain variable regionsequence; E, mouse immunoglobulin heavy chain enhancer sequence; L_(κ),mouse immunoglobulin light chain signal sequence; φ5_(κ), mouseimmunoglobulin light chain variable region sequence; Neo^(R) , E. colineomycin resistance gene.

FIG. 10 illustrates the constructions Zem229R, pφ5V_(H)Fab-neo and pWKI.Symbols used are set forth in FIG. 9.

FIGS. 11A-11D illustrate the sequence of a representative PDGFα-receptor cDNA and the deduced amino acid sequence (using standardone-letter codes) encoded by the cDNA and correspond to Sequence IDNumbers 35 and 36. Numbers at the ends of the lines refer to nucleotidepositions. Numbers below the sequence refer to amino acid positions.

FIG. 12 illustrates the assembly of a cDNA molecule encoding a PDGFα-receptor. Complementary DNA sequences are shown as lines. Only thoseportions of the vectors adjacent to the cDNA inserts are shown.

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention, it may be helpful to anunderstanding thereof to set forth definitions of certain terms to beused hereinafter.

DNA Construct: A DNA molecule, or a clone of such a molecule, eithersingle- or double-stranded that has been modified through humanintervention to contain segments of DNA combined and juxtaposed in amanner that as a whole would not otherwise exist in nature.

DNA constructs contain the information necessary to direct theexpression and/or secretion of DNA sequences encoding polypeptides ofinterest. DNA constructs will generally include promoters, enhancers andtranscription terminators. DNA constructs containing the informationnecessary to direct the secretion of a polypeptide will also contain atleast one secretory signal sequence.

Secretory Signal Sequence: A DNA sequence encoding a secretory peptide.A secretory peptide is an amino acid sequence that acts to direct thesecretion of a mature polypeptide or protein from a cell. Secretorypeptides are characterized by a core of hydrophobic amino acids and aretypically (but not exclusively) found at the amino termini of newlysynthesized proteins. Very often the secretory peptide is cleaved, fromthe mature protein during secretion. Such secretory peptides containprocessing sites that allow cleavage of the signal peptides from themature proteins as it passes through the secretory pathway. Processingsites may be encoded within the signal peptide or may be added to thesignal peptide by, for example, in vitro mutagenesis. Certain secretorypeptides may be used in concert to direct the secretion of polypeptidesand proteins. One such secretory peptide that may be used in combinationwith other secretory peptides is the third domain of the yeast Barrierprotein.

Receptor Analog: A non-immunoglobulin polypeptide comprising a portionof a receptor which is capable of binding ligand and/or is recognized byanti-receptor antibodies. The amino acid sequence of the receptor analogmay contain additions, substitutions or deletions as compared to thenative receptor sequence. A receptor analog may be, for example, theligand-binding domain of a receptor joined to another protein.Platelet-derived growth factor receptor (PDGF-R) analogs may, forexample, comprise a portion of a PDGF receptor capable of bindinganti-PDGF receptor antibodies, PDGF, PDGF isoforms, PDGF analogs, orPDGF antagonists.

Dimerizing Protein: A polypeptide chain having affinity for a secondpolypeptide chain, such that the two chains associate underphysiological conditions to form a dimer. The second polypeptide chainmay be the same or a different chain.

Biological activity: A function or set of activities performed by amolecule in a biological context (i.e., in an organism or an in vitrofacsimile thereof). Biological activities may include the induction ofextracellular matrix secretion from responsive cell lines, the inductionof hormone secretion, the induction of chemotaxis, the induction ofmitogenesis, the induction of differentiation, or the inhibition of celldivision of responsive cells. A recombinant protein or peptide isconsidered to be biologically active if it exhibits one or morebiological activities of its native counterpart.

Ligand: A molecule capable of being bound by the ligand-binding domainof a receptor or by a receptor analog. The molecule may be chemicallysynthesized or may occur in nature. Ligands may be grouped into agonistsand antagonists. Agonists are those molecules whose binding to areceptor induces the response pathway within a cell. Antagonists arethose molecules whose binding to a receptor blocks the response pathwaywithin a cell.

Joined: Two or more DNA coding sequences are said to be joined when, asa result of in-frame fusions between the DNA coding sequences or as aresult of the removal of intervening sequences by normal cellularprocessing, the DNA coding sequences are translated into a polypeptidefusion.

As noted above, the present invention provides methods for producingbiologically active dimerized polypeptide fusions and secreted receptoranalogs, which include, for example, PDGF receptor analogs. Secretedreceptor analogs may be used to screen for new compounds that act asagonists or antagonists when interacting with cells containingmembrane-bound receptors. In addition, the methods of the presentinvention provide dimerized non-immunoglobulin polypeptide fusions oftherapeutic value that are biologically active only as dimers. Moreover,the present invention provides methods of producing polypeptide dimersthat are biologically active only as non-covalently associated dimers.Secreted, biologically active dimers that may be produced using thepresent invention include nerve growth factor, colony stimulatingfactor-1, factor XIII, and transforming growth factor β.

As used herein, the ligand-binding domain of a receptor is that portionof the receptor that is involved with binding the natural ligand. Whilenot wishing to be bound by theory, the binding of a natural ligand to areceptor is believed to induce a conformational change which elicits aresponse to the change within the response pathway of the cell. Formembrane-bound receptors, the ligand-binding domain is generallybelieved to comprise the extracellular domain for the receptor. Thestructure of receptors may be predicted from the primary translationproducts using the hydrophobicity plot function of, for example, P/CGene or Intelligenetics Suite (Intelligenetics, Mt. View, CA) or may bepredicted according to the methods described, for example, by Kyte andDoolittle, J. Mol. Biol. 157:105-132, 1982. The ligand-binding domain ofthe PDGF β-receptor, for example, has been predicted to include aminoacids 29-531 of the published sequence (Gronwald et al., ibid.). Theligand-binding domain of the PDGF β-receptor has been predicted toinclude amino acids 25-500 of the published α-receptor sequence (Matsuiet al., ibid.). As used herein, the ligand-binding domain of the PDGFβ-receptor includes amino acids 29-441 of the sequence of FIGS. 1A-1G(Sequence ID Number 1) and C-terminal extensions up to and includingamino acid 531. The ligand-binding domain of the PDGF α-receptor isunderstood to include amino acids 24-524 of FIGS. 11A-11D (Sequence IDNumbers 35 and 36).

Receptor analogs that may be used in the present invention include theligand-binding domains of the epidermal growth factor receptor (EGF-R)and the insulin receptor. As used herein, a ligand-binding domain isthat portion of the receptor that is involved in binding ligand and isgenerally a portion or essentially all of the extracellular domain thatextends from the plasma membrane into the extracellular space. Theligand-binding domain of the EGF-R, for example, resides in theextracellular domain. EGF-R dimers have been found to exhibit higherligand-binding affinity than EGF-R monomers (Boni-Schnetzler and Pilch,Proc. Natl. Acad. Sci. USA 84:7832-7836, 1987). The insulin receptor(Ullrich et al., Nature 313:756-761, 1985) requires dimerization forbiological activity.

Another example of a receptor that may be secreted from a host cell is aplatelet-derived growth factor receptor (PDGF-R). Two classes ofPDGF-Rs, which recognized different isoforms of PDGF, have beenidentified. (PDGF is a disulfide-bonded, two-chain molecule, which ismade up of an A chain and a B chain. These chains may be combined as ABheterodimers, AA homodimers or BB homodimers. These dimeric moleculesare referred to herein as “isoforms”.) The β-receptor (PDGFβ-R), whichrecognizes only the BB isoform of PDGF (PDGF-BB), has been described(Claesson-Welsh et al., Mol. Cell. Biol. 8:3476-3486, 1988; Gronwald etal., Proc. Natl. Acad. Sci. USA 85:3435-3439, 1988). The α-receptor(PDGFα-R), which recognizes all three PDGF isoforms (PDGF-AA, PDGF-ABand PDGF-BB), has been described by Matsui et al. (Science 243:800-804,1989) and Kelly and Murray (pending commonly assigned U.S. patentapplication Ser. No. 07/355,018 now abandoned which is incorporatedherein by reference). The primary translation products of thesereceptors indicate that each includes an extracellular domain implicatedin the ligand-binding process, a transmembrane domain, and a cytoplasmicdomain containing a tyrosine kinase activity.

The present invention provides a standardized assay system, notpreviously available in the art, for determining the presence of PDGF,PDGF isoforms, PDGF agonists or PDGF antagonists using a secreted PDGFreceptor analogs. Such an assay system will typically involve combiningthe secreted PDGF receptor analog with a biological sample underphysiological conditions which permit the formation of receptor-ligandcomplexes, followed by detecting the presence of the receptor-ligandcomplexes. The term physiological conditions is meant to include thoseconditions found within the host organism and include, for example, theconditions of osmolarity, salinity and pH. Detection may be achievedthrough the use of a label attached to the PDGF receptor analog orthrough the use of a labeled antibody which is reactive with thereceptor analog or the ligand. A wide variety of labels may be utilized,such as radionuclides, fluorophores, enzymes and luminescers.Receptor-ligand complexes may also be detected visually, i.e., inimmunoprecipitation assays which do not require the use of a label. Thisassay system provides secreted PDGF receptor analogs that may beutilized in a variety of screening assays for, for example, screeningfor analogs of PDGF. The present invention also provides a methods formeasuring the level of PDGF and PDGF isoforms in biological fluids.

As noted above, the present invention provides methods for producingdimerized polypeptide fusions that require dimerization for biologicalactivity or enhancement of biological activity. Polypeptides requiringdimerization for biological activity include, in addition to certainreceptors, nerve growth factor, colony-stimulating factor-1 (CSF-1),transforming growth factor β (TGF-β), PDGF, and factor XIII. Nervegrowth factor is a non-covalently linked dimer (Harper et al., J. Biol.Chem. 257: 8541-8548, 1982). CSF-1, which specifically stimulates theproliferation and differentiation of cells of mononuclear phagocyticlineage, is a disulfide-bonded homodimer (Retternmier et al., Mol. Cell.Biol. 7: 2378-2387, 1987). TGF-β is biologically active as adisulfide-bonded dimer (Assoian et al., J. Biol. Chem. 258: 7155-7160,1983). Factor XIII is a plasma protein that exists as a two chainhomodimer in its activated form (Ichinose et al., Biochem. 25:6900-6906, 1986). PDGF, as noted above, is a disulfide-bonded, two chainmolecule (Murray et al., U.S. Pat. No. 4,766,073).

The present invention provides methods by which receptor analogs,including receptor analogs and PDGF-R analogs, requiring dimerizationfor activity may be secreted from host cells. The methods describedherein are particularly advantageous in that they allow the productionof large quantities of purified receptors. The receptors may be used inassays for the screening of potential ligands, in assays for bindingstudies, as imaging agents, and as agonists and antagonists withintherapeutic agents.

A DNA sequence encoding a human PDGF receptor may be isolated as a cDNAusing techniques known in the art (see, for example, Okayama and Berg,Mol. Cell. Biol. 2: 161-170, 1982; Mol. Cell. Biol. 3: 280-289, 1983)from a library of human genomic or cDNA sequences. Such libraries may beprepared by standard procedures, such as those disclosed by Gubler andHoffman (Gene 25: 263-269, 1983). It is preferred that the molecule is acDNA molecule because cDNA lack introns and are therefore more suited tomanipulation and expression in transfected or transformed cells. Sourcesof mRNA for use in the preparation of a cDNA library include the MG-63human osteosarcoma cell line (available from ATCC under accession numberCRL 1427), diploid human dermal fibroblasts and human embryo fibroblastand brain cells (Matsui et al., ibid.). A cDNA encoding a PDGFβ-R hasbeen cloned from a diploid human dermal fibroblast cDNA library usingoligonucleotide probes complementary to sequences of the mouse PDGFreceptor (Gronwald et al., ibid.). A PDGFα-R cDNA has been isolated byMatsui et al. (ibid.) from human embryo fibroblast and brain cells.Alternatively, a cDNA encoding a PDGFα-R may be isolated from a libraryprepared from MG-63 human osteosarcoma cells using a cDNA probecontaining sequences encoding the transmembrane and cytoplasmic domainsof the PDGFβ-R (described by Kelly and Murray, ibid.). Partial cDNAclones (fragments) can be extended by re-screening of the library withthe cloned cDNA fragment until the full sequence is obtained. In oneembodiment, a ligand-binding domain of a PDGF receptor is encoded by thesequence of FIGS. 1A-1G (Sequence ID Number 1) from amino acid 29through amino acid 441. In another embodiment, a ligand-binding domainof a PDGF receptor is encoded by the sequence of FIGS. 1A-1G (SequenceID Number 1) from amino acid 29 through amino acid 531. In yet anotherembodiment, a ligand-binding domain of a PDGF receptor is encoded by thesequence of FIGS. 11A-11D (Sequence ID Numbers 35 and 36) from aminoacid 24 through amino acid 524. One skilled in the art may envision theuse of a smaller DNA sequence encoding the ligand-binding domain of aPDGF receptor containing at least 400 amino acids of the extracellulardomain.

DNA sequences encoding EGF-R (Ullrich et al., Nature 304: 418-425,1984), the insulin receptor (Ullrich et al., Nature 313: 756-761, 1985),nerve growth factor (Ullrich et al. Nature 303: 821-825, 1983), colonystimulating factor-1 (Rettenmier et al., ibid.), transforming growthfactor β (Derynck et al., Nature 316: 701-705, 1985), PDGF (Murray etal., ibid.), and factor XIII (Ichinose et al., ibid.) may also be usedwithin the present invention.

To direct polypeptides requiring dimerization for biological activity orreceptor analogs into the secretory pathway of the host cell, at leastone secretory signal sequence is used in conjunction with the DNAsequence of interest. Preferred secretory signals include the alphafactor signal sequence (pre-pro sequence) (Kurjan and Herkowitz, Cell30: 933-943, 1982; Kurjan et al., U.S. Pat. No. 4,546,082; Brake, EP116,201, 1983), the PHO5 signal sequence (Beck et al., WO 86/00637), theBAR1 secretory signal sequence (MacKay et al., U.S. Pat. No. 4,613,572;MacKay, WO 87/002670), immunoglobulin V_(H) signal sequences (Loh etal., Cell 23: 85-93, 1983; Watson Nuc. Acids. Res. 12: 5145-5164, 1984)and immunoglobulin V_(κ) signal sequences (Watson, ibid.). Particularlypreferred signal sequences are the SUC2 signal sequence (Carlson et al.,Mol. Cell. Biol. 3: 439-447, 1983) and PDGF receptor signal sequences.Alternatively, secretory signal sequences may be synthesized accordingto the rules established, for example, by von Heinje (Eur. J. Biochem.133: 17-21, 1983; J. Mol. Biol. 134: 99-105, 1985; Nuc. Acids. Res. 14:4683-3690, 1986).

Secretory signal sequences may be used singly or may be combined. Forexample, a first secretory signal sequence may be used singly orcombined with a sequence encoding the third domain of Barrier (describedin co-pending commonly assigned U.S. patent application Ser. No.07/104,316 now abandoned, which is incorporated by reference herein inits entirety). The third domain of Barrier may be positioned in properreading frame 3′ of the DNA sequence of interest or 5′ to the DNAsequence and in proper reading frame with both the secretory signalsequence and the DNA sequence of interest.

In one embodiment of the present invention, a sequence encoding adimerizing protein is joined to a sequence encoding a polypeptide chainof a polypeptide dimer or a receptor analog, and this fused sequence isjoined in proper reading frame to a secretory signal sequence. As shownherein, the present invention utilizes such an arrangement to drive theassociation of the polypeptide or receptor analog to form a biologicallyactive molecule upon secretion. Suitable dimerizing proteins include theS. cerevisiae repressible acid phosphatase (Mizunaga et al., J. Biochem.(Tokyo) 103: 321-326, 1988), the S. cerevisiae type 1 killer preprotoxin(Sturley et al., EMBO J. 5: 3381-3390, 1986), the S. calsbergensis alphagalactosidase melibiase (Sumner-Smith et al., Gene 36: 333-340, 1985),the S. cerevisiae invertase (Carlson et al., Mol. Cell. Biol. 3:439-447, 1983), the Neurospora crassa ornithine decarboxylase (Digangiet al., J. Biol. Chem. 262: 7889-7893, 1987), immunoglobulin heavy chainhinge regions (Takahashi et al., Cell 29: 671-679, 1982), and otherdimerizing immunoglobulin sequences. In a preferred embodiment, S.cerevisiae invertase is used to drive the association of polypeptidesinto dimers. Portions of dimerizing proteins, such as those mentionedabove, may be used as dimerizing proteins where those portions arecapable of associating as a dimer in a covalent or noncovlent manner.Such portions may be determined by, for example, altering a sequenceencoding a dimerizing protein through in vitro mutagenesis to deleteportions of the coding sequence. These deletion mutants may be expressedin the appropriate host to determine which portions retain thecapability of associating as dimers. Portions of immunoglobulin genesequences may be used to drive the association of non-immunoglobulinpolypeptides. These portions correspond to discrete domains ofimmunoglobulins. Immunoglobulins comprise variable and constant regions,which in turn comprise discrete domains that show similarity in theirthree-dimensional conformations. These discrete domains correspond toimmunoglobulin heavy chain constant region domain exons, immunoglobulinheavy chain variable region domain exons, immunoglobulin light chainvariable region domain exons and immunoglobulin light chain constantregion domain exons in immunoglobulin genes (Hood et al., in Immunology,The Benjamin/Cummings Publishing Company, Inc., Menlo Park, Calif.;Honjo et al., Cell 18: 559-568, 1979; Takahashi et al., Cell 29:671-679, 1982; and Honjo, Ann. Rev. Immun. 1:499-528, 1983)).Particularly preferred portions of immunoglobulin heavy chains includeFab and Fab′ fragments. (An Fab fragment is a portion of animmunoglobulin heavy chain that includes a heavy chain variable regiondomain and a heavy chain constant region domain. An Fab′ fragment is aportion of an immunoglobulin heavy chain that includes a heavy chainvariable region domain, a heavy chain constant region domain and a heavychain hinge region domain.)

It is preferred to use an immunoglobulin light chain constant region inassociation with at least one immunoglobulin heavy chain constant regiondomain. In another embodiment, an immunoglobulin light chain constantregion is associated with at least one immunoglobulin heavy chainconstant region domain joined to an immunoglobulin hinge region. In oneset of embodiments, an immunoglobulin light chain constant region joinedin frame with a polypeptide chain of a non-immunoglobulin polypeptidedimer or receptor analog and is associated with at least one heavy chainconstant region. In a preferred set of embodiments a variable region isjoined upstream of and in proper reading frame with at least oneimmunoglobulin heavy chain constant: region. In another set ofembodiments, an immunoglobulin heavy chain is joined in frame with apolypeptide chain of a non-immunoglobulin polypeptide dimer or receptoranalog and is associated with an immunoglobulin light chain constantregion. In yet another set of embodiments, a polypeptide chain of anon-immunoglobulin polypeptide dimer or receptor analog is joined to atleast one immunoglobulin heavy chain constant region which is joined toan immunoglobulin hinge region and is associated with an immunoglobulinlight chain constant region. In a preferred set of embodiments animmunoglobulin variable region is joined upstream of and in properreading frame with the immunoglobulin light chain constant region.

Immunoglobulin heavy chain constant region domains include C_(H)1,C_(H)2, C_(H)3, and C_(H)4 of any class of immunoglobulin heavy chainincluding γ, α, ε, μ, and δ classes (Honjo, ibid., 1983) A particularlypreferred immunoglobulin heavy chain constant region domain is humanC_(H)1. Immunoglobulin variable regions include V_(H), V_(κ), or V_(λ).

DNA sequences encoding immunoglobulins may be cloned from a variety ofgenomic or cDNA libraries known in the art. The techniques for isolatingsuch DNA sequences using probe-based methods are conventional techniquesand are well known to those skilled in the art. Probes for isolatingsuch DNA sequences may be based on published DNA sequences (see, forexample, Hieter et al., Cell 22: 197-207, 1980). Alternatively, thepolymerase chain reaction (PCR) method disclosed by Mullis et al. (U.S.Pat. No. 4,683,195) and Mullis (U.S. Pat. No. 4,683,202), incorporatedherein by reference may be used. The choice of library and selection ofprobes for the isolation of such DNA sequences is within the level ofordinary skill in the art.

Host cells for use in practicing the present invention includeeukaryotic cells capable of being transformed or transfected withexogenous DNA and grown in culture, such as cultured mammalian andfungal cells. Fungal cells, including species of yeast (e.g.,Saccharomyces spp., Schizosaccharomyces spp.), or filamentous fungi(e.g., Aspergillus spp., Neurospora spp.) may be used as host cellswithin the present invention. Strains of the yeast Saccharomycescerevisiae are particularly preferred.

Expression units for use in the present invention will generallycomprise the following elements, operably linked in a 5′ to 3′orientation: a transcriptional promoter, a secretory signal sequence aDNA sequence encoding nonimmunoglobulin polypeptide requiringdimerization for biological activity joined to a dimerizing protein anda transcriptional terminator. The selection of suitable promoters,signal sequences and terminators will be determined by the selected hostcell and will be evident to one skilled in the art and are discussedmore specifically below.

Suitable yeast vectors for use in the present invention include YRp7(Struhl et al., Proc. Natl. Acad. Sci. USA 76: 1035-1039, 1978), YEp13(Broach et al., Gene 8: 121-133, 1979), pJDB249 and pJDB219 (Beggs,Nature 275:104-108, 1978) and derivatives thereof. Such vectors willgenerally include a selectable marker, which may be one of any number ofgenes that exhibit a dominant phenotype for which a phenotypic assayexists to enable transformants to be selected. Preferred selectablemarkers are those that complement host cell auxotrophy, provideantibiotic resistance or enable a cell to utilize specific carbonsources, and include LEU2 (Broach et al. ibid.), URA3 (Botstein et al.,Gene 8: 17, 1979), HIS3 (Struhl et al., ibid.) or POT1 (Kawasaki andBell, EP 171,142). Other suitable selectable markers include the CATgene, which confers chloramphenicol resistance on yeast cells.

Preferred promoters for use in yeast include promoters from yeastglycolytic genes (Hitzeman et al., J. Biol. Chem. 255: 12073-12080,1980; Alber and Kawasaki, J. Mol. Appl. Genet. 1: 419-434, 1982;Kawasaki, U.S. Pat. No. 4,599,311) or alcohol dehydrogenase genes (Younget al., in Genetic Engineering of Microorganisms for Chemicals,Hollaender et al., (eds.), p. 355, Plenum, New York, 1982; Ammerer,Meth. Enzymol. 101: 192-201, 1983). In this regard, particularlypreferred promoters are the TPI1 promoter (Kawasaki, U.S. Pat. No.4,599,311, 1986) and the ADH2-4^(c) promoter (Russell et al., Nature304: 652-654, 1983 and Irani and Kilgore, described in pending, commonlyassigned U.S. patent application Ser. No. 07/183,130, which isincorporated herein by reference). The expression units may also includea transcriptional terminator. A preferred transcriptional terminator isthe TPI1 terminator (Alber and Kawasaki, ibid.).

In addition to yeast, proteins of the present invention can be expressedin filamentous fungi, for example, strains of the fungi Aspergillus(McKnight and Upshall, described in commonly assigned U.S. Pat. No.4,935,349, which is incorporated herein by reference). Examples ofuseful promoters include those derived from Aspergillus nidulansglycolytic genes, such as the ADH3 promoter (McKnight et al., EMBO J. 4:2093-2099, 1985) and the tpiA promoter. An example of a suitableterminator is the ADH3 terminator (McKnight et al., ibid.). Theexpression units utilizing such components are cloned into vectors thatare capable of insertion into the chromosomal DNA of Aspergillus.

Techniques for transforming fungi are well known in the literature, andhave been described, for instance, by Beggs (ibid.), Hinnen et al.(Proc. Natl. Acad. Sci. USA 75: 1929-1933, 1978), Yelton et al., (Proc.Natl. Acad. Sci. USA 81: 1740-1747, 1984), and Russell (Nature 301.167-169, 1983). The genotype of the host cell will generally contain agenetic defect that is complemented by the selectable marker present onthe expression vector. Choice of a particular host and selectable markeris well within the level of ordinary skill in the art.

In a preferred embodiment, a Saccharomyces cerevisiae host cell thatcontains a genetic deficiency in a gene required for asparagine-linkedglycosylation of glycoproteins is used. Preferably, the S. cerevisiaehost cell contains a genetic deficiency in the MNN9 gene (described inpending, commonly assigned U.S. patent application Ser. Nos. 116,095 and189,547 which are incorporated by reference herein in their entirety).Most preferably, the S. cerevisiae host cell contains a disruption ofthe MNN9 gene. S. cerevisiae host cells having such defects may beprepared using standard techniques of mutation and selection. Ballou etal. (J. Biol. Chem. 255: 5986-5991, 1980) have described the isolationof mannoprotein biosynthesis mutants that are defective in genes whichaffect asparagine-linked glycosylation. Briefly, mutagenized S.cerevisiae cells were screened using fluoresceinated antibodies directedagainst the outer mannose chains present on wild-type yeast. Mutantcells that did not bind antibody were further characterized and werefound to be defective in the addition of asparagine-linkedoligosaccharide moieties. To optimize production of the heterologousproteins, it is preferred that the host strain carries a mutation, suchas the S. cerevisiae pep4 mutation (Jones, Genetics 85: 23-33, 1977),which results in reduced proteolytic activity.

In addition to fungal cells, cultured mammalian cells may be used ashost cells within the present invention. Preferred cell lines are rodentmyeloma cell lines, which include p3X63Ag8 (ATCC TIB 9), FO (ATCC CRL1646), NS-1 (ATCC TIB 18) and 210-RCY-Ag1 (Galfre et al., Nature 277:131, 1979). A particularly preferred rodent myeloma cell line isSP2/0-Ag14 (ATCC CRL 1581). In addition, a number of other cell linesmay be used within the present invention, including COS-1 (ATCC CRL1650), BHK, p363.Ag.8.653 (ATCC CRL 1580) Rat Hep I (ATCC CRL 1600), RatHep II (ATCC CRL 1548), TCMK (ATCC CCL 139), Human lung (ATCC CCL 75.1),Human hepatoma (ATCC HTB-52), Hep G2 (ATCC HB 8065), Mouse liver (ATCCCC 29.1), 293 (ATCC CRL 1573; Graham et al., J. Gen. Virol. 36: 59-72,1977) and DUKX cells (Urlaub and Chasin, Proc. Natl. Acad. Sci USA 77:4216-4220, 1980) A preferred BHK cell line is the tk⁻ts13 BHK cell line(Waechter and Baserga, Proc. Natl. Acad. Sci USA 79: 1106-1110, 1982). Apreferred BHK cell line is the tk⁻ts13 BHK cell line (Waechter andBaserga, Proc. Natl. Acad. Sci. USA 79: 1106-1110, 1982). A tk⁻ BHK cellline is available from the American Type Culture Collection, Rockville,Md., under accession number CRL 1632. A particularly preferred tk⁻ BHKcell line is BHK 570 which is available from the American Type CultureCollection under accession number 10314.

Mammalian expression vectors for use in carrying out the presentinvention will include a promoter capable of directing the transcriptionof a cloned gene or cDNA. Preferred promoters include viral promotersand cellular promoters. Preferred viral promoters include the major latepromoter from adenovirus 2 (Kaufman and Sharp, Mol. Cell. Biol. 2:1304-13199, 1982) and the SV40 promoter (Subramani et al., Mol. Cell.Biol. 1: 854-864, 1981). Preferred cellular promoters include the mousemetallothionein 1 promoter (Palmiter et al., Science 222: 809-814, 1983)and a mouse V_(κ) promoter (Grant et al., Nuc. Acids Res. 15: 5496,1987). A particularly preferred promoter is a mouse V_(H) promoter (Lohet al., ibid.). Such expression vectors may also contain a set of RNAsplice sites located downstream from the promoter and upstream from theDNA sequence encoding the peptide or protein of interest. Preferred RNAsplice sites may be obtained from adenovirus and/or immunoglobulingenes. Also contained in the expression vectors is a polyadenylationsignal located downstream of the coding sequence of interest.Polyadenylation signals include the early or late polyadenylationsignals from SV40 (Kaufman and Sharp, ibid.), the polyadenylation signalfrom the adenovirus 5 E1B region and the human growth hormone geneterminator (DeNoto et al., Nuc. Acids Res. 9: 3719-3730, 1981). Aparticularly preferred polyadenylation signal is the V_(H) geneterminator (Loh et al., ibid.). The expression vectors may include anoncoding viral leader sequence, such as the adenovirus 2 tripartiteleader, located between the promoter and the RNA splice sites. Preferredvectors may also include enhancer sequences, such as the SV40 enhancerand the mouse μ enhancer (Gillies, Cell 33: 717-728, 1983). Expressionvectors may also include sequences encoding the adenovirus VA RNAs.

Cloned DNA sequences may be introduced into cultured mammalian cells by,for example, calcium phosphate-mediated transfection (Wigler et al.,Cell 14: 725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7: 603,1981; Graham and Van der Eb, Virology 52: 456, 1973.) Other techniquesfor introducing cloned DNA sequences into mammalian cells, such aselectroporation (Neumann et al., EMBO J. 1: 841-845, 1982), may also beused. In order to identify cells that have integrated the cloned DNA, aselectable marker is generally introduced into the cells along with thegene or cDNA of interest. Preferred selectable markers for use incultured mammalian cells include genes that confer resistance to drugs,such as neomycin, hygromycin, and methotrexate. The selectable markermay be an amplifiable selectable marker. A preferred amplifiableselectable marker is the DHFR gene. A particularly preferred amplifiablemarker is the DHFR^(r) cDNA (Simonsen and Levinson, Proc. Natl. Acad.Sci. USA 80: 2495-2499, 1983). Selectable markers are reviewed by Thilly(Mammalian Cell Technology, Butterworth Publishers, Stoneham, Mass.) andthe choice of selectable markers is well within the level of ordinaryskill in the art.

Selectable markers may be introduced into the cell on a separate plasmidat the same time as the gene of interest, or they may be introduced onthe same plasmid. If on the same plasmid, the selectable marker and thegene of interest may be under the control of different promoters or thesame promoter, the latter arrangement producing a dicistronic message.Constructs of this type are known in the art (for example, Levinson andSimonsen, U.S. Pat. No. 4,713,339). It may also be advantageous to addadditional DNA, known as “carrier DNA” to the mixture which isintroduced into the cells.

Transfected mammalian cells are allowed to grow for a period of time,typically 1-2 days, to begin expressing the DNA sequence(s) of interest.Drug selection is then applied to select for growth of cells that areexpressing the selectable marker in a stable fashion. For cells thathave been transfected with an amplifiable selectable marker the drugconcentration may be increased in a stepwise manner two select forincreased copy number of the cloned sequences, thereby increasingexpression levels.

Host cells containing DNA constructs of the present invention are grownin an appropriate growth medium. As used herein, the term “appropriategrowth medium” means a medium containing nutrients required for thegrowth of cells. Nutrients required for cell growth may include a carbonsource, a nitrogen source, essential amino acids, vitamins, minerals andgrowth factors. The growth medium will generally select for cellscontaining the DNA construct by, for example, drug selection ordeficiency in an essential nutrient which are complemented by theselectable marker on the DNA construct or co-transfected with the DNAconstruct. Yeast cells, for example, are preferably grown in achemically defined medium, comprising a non-amino acid nitrogen source,inorganic salts, vitamins and essential amino acid supplements. The pHof the medium is preferably maintained at a pH greater than 2 and lessthan 8, preferably at pH 6.5. Methods for maintaining a stable pHinclude buffering and constant pH control, preferably through theaddition of sodium hydroxide. Preferred buffering agents includesuccinic acid and Bis-Tris (Sigma Chemical Co., St. Louis, Mo.). Yeastcells having a defect in a gene required for asparagine-linkedglycosylation are preferably grown in a medium containing an osmoticstabilizer. A preferred osmotic stabilizer is sorbitol supplemented intothe medium at a concentration between 0.1 M and 1.5 M., preferably at0.5 M or 1.0 M. Cultured mammalian cells are generally grown incommercially available serum-containing or serum-free media. Selectionof a medium appropriate for the particular cell line used is within thelevel of ordinary skill in the art.

The culture medium from appropriately grown transformed or transfectedhost cells is separated from the cell material, and the presence ofdimerized polypeptide fusions or secreted receptor analogs isdemonstrated. A preferred method of detecting receptor analogs, forexample, is by the binding of the receptors or portions of receptors toa receptor-specific antibody. An anti-receptor antibody may be amonoclonal or polyclonal antibody raised against the receptor inquestion, for example, an anti-PDGF receptor monoclonal antibody may beused to assay for the presence of PDGF receptor analogs. Anotherantibody, which may be used for detecting substance P-tagged peptidesand proteins, is a commercially available rat anti-substance Pmonoclonal antibody which may be utilized to visualize peptides orproteins that are fused to the C-terminal amino acids of substance P.Ligand binding assays may also be used to detect the presence ofreceptor analogs. In the case of PDGF receptor analogs, it is preferableto use fetal foreskin fibroblasts, which express PDGF receptors, tocompete against the PDGF receptor analogs of the present invention forlabeled PDGF and PDGF isoforms.

Assays for detection of secreted, biologically active peptide dimers andreceptor analogs may include Western transfer, protein blot or colonyfilter. A Western transfer filter may be prepared using the methoddescribed by Towbin et al. (Proc. Natl. Acad. Sci. USA 76: 4350-4354,1979). Briefly, samples are electrophoresed in a sodium dodecylsulfatepolyacrylamide gel. The proteins in the gel are electrophoreticallytransferred to nitrocellulose paper. Protein blot filters may beprepared by filtering supernatant samples or concentrates throughnitrocellulose filters using, for example, a Minifold (Schleicher &Schuell, Keene, N.H.). Colony filters may be prepared by growingcolonies on a nitrocellulose filter that has been laid across anappropriate growth medium. In this method, a solid medium is preferred.The cells are allowed to grow on the filters for at least 12 hours. Thecells are removed from the filters by washing with an appropriate bufferthat does not remove the proteins bound to the filters. A preferredbuffer comprises 25 mM Tris-base, 19 mM glycine, pH 8.3, 20% methanol.

The dimerized polypeptide fusions and receptor analogs present on theWestern transfer, protein blot or colony filters may be visualized byspecific antibody binding using methods known in the art. For example,Towbin et al. (ibid.) describe the visualization of proteins immobilizedon nitrocellulose filters with a specific antibody followed by a labeledsecond antibody, directed against the first antibody. Kits and reagentsrequired for visualization are commercially available, for example, fromVector Laboratories, (Burlingame, Calif.), and Sigma Chemical Company(St. Louis, Mo.).

Secreted, biologically active dimerized polypeptide fusions and receptoranalogs may be isolated from the medium of host cells grown underconditions that allow the secretion of the biologically active dimerizedpolypeptide fusions and receptor analogs. The cell material is removedfrom the culture medium, and the biologically active dimerizedpolypeptide fusions and receptor analogs are isolated using isolationtechniques known in the art. Suitable isolation techniques includeprecipitation and fractionation by a variety of chromatographic methods,including gel filtration, ion exchange chromatography and immunoaffinitychromatography. A particularly preferred purification method isimmunoaffinity chromatography using an antibody directed against thereceptor analog or dimerized polypeptide fusion. The antibody ispreferably immobilized or attached to a solid support or substrate. Aparticularly preferred substrate is CNBr-activated Sepharose (PharmaciaLKB Technologies, Inc., Piscataway, N.J.). By this method, the medium iscombined with the antibody/substrate under conditions that will allowbinding to occur. The complex may be washed to remove unbound material,and the receptor analog or peptide dimer is released or eluted throughthe use of conditions unfavorable to complex formation. Particularlyuseful methods of elution include changes in pH, wherein the immobilizedantibody has a high affinity for the ligand at a first pH and a reducedaffinity at a second (higher or lower) pH; changes in concentration ofcertain chaotropic agents; or through the use of detergents.

The secreted PDGF receptor analogs of the present invention can be usedwithin a variety of assays for detecting the presence of and/orscreening for native PDGF, PDGF isoforms or PDGF-like molecules. Theseassays will typically involve combining PDGF receptor analogs, which maybe bound to a solid substrate such as polymeric microtiter plate wells,with a biological sample under conditions that permit the formation ofreceptor/ligand complexes. Screening assays for the detection of PDGF,PDGF isoforms or PDGF-like molecules will typically involve combiningsoluble PDGF receptor analogs with a biological sample and incubatingthe mixture with a PDGF isoform or mixture of PDGF isoforms bound to asolid substrate such as polymeric microtiter plates, under conditionsthat permit the formation of receptor/ligand complexes. Detection may beachieved through the use of a label attached to the receptor or throughthe use of a labeled antibody which is reactive with the receptor.Alternatively, the labeled antibody may be reactive with the ligand. Awide variety of labels may be utilized, such as radionuclides,fluorophores, enzymes and luminescers. Complexes may also be detectedvisually, i.e., in immunoprecipitation assays, which do not require theuse of a label.

Secreted PDGF receptor analogs of the present invention may also belabeled with a radioisotope or other imaging agent and used for in vivodiagnostic purposes. Preferred radioisotope imaging agents includeiodine-125 and technetium-99, with technetium-99 being particularlypreferred. Methods for producing protein-isotope conjugates are wellknown in the art, and are described by, for example, Eckelman et al.(U.S. Pat. No. 4,652,440), Parker et al. (WO 87/05030) and Wilber et al.(EP 203,764). Alternatively, the secreted receptor analogs may be boundto spin label enhancers and used for magnetic resonance (MR) imaging.Suitable spin label enhancers include stable, sterically hindered, freeradical compounds such as nitroxides. Methods for labeling ligands forMR imaging are disclosed by, for example, Coffman et al. (U.S. Pat. No.4,656,026). For administration, the labeled receptor analogs arecombined with a pharmaceutically acceptable carrier or diluent, such assterile saline or sterile water. Administration is preferably by bolusinjection, preferably intravenously. These imaging agents areparticularly useful in identifying the locations of atheroscleroticplaques, PDGF-producing tumors, and receptor-bound PDGF.

The secreted PDGF receptor analogs of the present invention may also beutilized within diagnostic kits. Briefly, the subject receptor analogsare preferably provided in a lyophilized form or immobilized onto thewalls of a suitable container, either alone or in conjunction withantibodies capable of binding to native PDGF or selected PDGF isoform(s)or specific ligands. The antibodies, which may be conjugated to a labelor unconjugated, are generally included in the kits with suitablebuffers, such as phosphate, stabilizers, inert proteins or the like.Generally, these materials are present in less than about 5% weightbased upon the amount of active receptor analog, and are usually presentin an amount of at least about 0.001% weight. It may also be desirableto include an inert excipient to dilute the active ingredients. Such anexcipient may be present from about 1% to 99% weight of the totalcomposition. In addition, the kits will typically include other standardreagents, instructions and, depending upon the nature of the labelinvolved, reactants that are required to produce a detectable product.Where an antibody capable of binding to the receptor or receptor/ligandcomplex is employed, this antibody will usually be provided in aseparate vial. The antibody is typically conjugated to a label andformulated in an analogous manner with the formulations brieflydescribed above. The diagnostic kits, including the containers, may beproduced and packaged using conventional kit manufacturing procedures.

As noted above, the secreted PDGF receptor analogs of the presentinvention may be utilized within methods for purifying PDGF from avariety of samples. Within a preferred method, the secreted PDGFreceptor analogs are immobilized or attached to a substrate or supportmaterial, such as polymeric tubes, beads, polysaccharide particulates,polysaccharide-containing materials, polyacrylamide or other waterinsoluble polymeric materials. Methods for immobilization are well knownin the art (Mosbach et al., U.S. Pat. No. 4,415,665; Clarke et al.,Meth. Enzymology 68: 436-442, 1979). A common method of immobilizationis CNBr activation. Activated substrates are commercially available froma number of suppliers, including Pharmacia (Piscataway, N.J.), PierceChemical Co. (Rockford, Ill.) and Bio-Rad Laboratories (Richmond,Calif.). A preferred substrate is CNBr-activated Sepharose (Pharmacia,Piscataway, N.J.). Generally, the substrate/receptor analog complex willbe in the form of a column. The sample is then combined with theimmobilized receptor analog under conditions that allow binding tooccur. The substrate with immobilized receptor analog is firstequilibrated with a buffer solution of a composition in which thereceptor analog has been previously found to bind its ligand. Thesample, in the solution used for equilibration, is then applied to thesubstrate/receptor analog complex. Where the complex is in the form of acolumn, it is preferred that the sample be passed over the column two ormore times to permit full binding of ligand to receptor analog. Thecomplex is then washed with the same solution to elute unbound material.In addition, a second wash solution may be used to minimize nonspecificbinding. The bound material may then be released or eluted through theuse of conditions unfavorable to complex formation. Particularly usefulmethods include changes in pH, wherein the immobilized receptor has ahigh affinity for PDGF at a first pH and reduced affinity at a second(higher or lower) pH; changes in concentration of certain chaotropicagents; or through the use of detergents.

The secreted PDGF receptor analogs fused to dimerizing proteins of thepresent invention may be used in pharmaceutical compositions for topicalor intravenous application. The secreted PDGF receptor analogs of thepresent invention may be useful in the treatment of atherosclerosis by,for example, binding endogenous PDGF to prevent smooth muscle cellproliferation. The PDGF receptor analogs fused to dimerizing proteinsare used in combination with a physiologically acceptable carrier ordiluent. Preferred carriers and diluents include saline and sterilewater. Pharmaceutical compositions may also contain stabilizers andadjuvants. The resulting aqueous solutions may be packaged for use orfiltered under aseptic conditions and lyophilized, the lyophilizedpreparation being combined with at sterile aqueous solution prior toadministration.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES

Enzymes, including restriction enzymes, DNA polymerase I (Klenowfragment), T4 DNA polymerase, T4 DNA ligase and T4 polynucleotidekinase, were obtained from New England Biolabs (Beverly, Mass.),GIBCO-BRL (Gaithersburg, Md.) and Boerhinger-Mannheim Biochemicals(Indianapolis, Ind.) and were used as directed by the manufacturer or asdescribed in Maniatis et al. (Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory, NY, 1982) and Sambrook et al. (MolecularCloning: A Laboratory Manual/Second Edition, Cold Spring HarborLaboratory, NY, 1989).

Example 1 Cloning PDGF Receptor cDNAs

A. Cloning the PDGF β-Receptor

A cDNA encoding the PDGF β-receptor was cloned as follows. ComplementaryDNA (cDNA) libraries were prepared from poly(A) RNA from diploid humandermal fibroblast cells, prepared by explant from a normal adult,essentially as described by Hagen et al. (Proc. Natl. Acad. Sci. USA 83:2412-2416, 1986). Briefly, the poly(A) RNA was primed with oligo d(T)and cloned into λgt11 using Eco RI linkers. The random primed librarywas screened for the presence of human PDGF receptor cDNA's using threeoligonucleotide probes complementary to sequences of the mouse PDGFreceptor (Yarden et al., Nature 323: 226-232, 1986). Approximately onemillion phage from the random primed human fibroblast cell library werescreened using oligonucleotides ZC904, ZC905 and. ZC-906 (Table 1;Sequence ID Numbers 5, 6 and 7, respectively). Eight positive cloneswere identified and plaque purified. Two clones, designated RP41 andRP51, were selected for further analysis by restriction enzyme mappingand DNA sequence analysis. RP51 was found to contain 356 bp of5′-noncoding sequence, the ATG translation initiation codon and 738 bpof the amino terminal coding sequence. RP41 was found to overlap cloneRP51 and contained 2649 bp encoding amino acids 43-925 of the β-receptorprotein.

TABLE 1 Oligonucleotide Sequendes ZC871 (Sequence ID Number 3) 5′ CTCTCT TCC TCA GGT AAA TGA GTG CCA GGG CCG GCA AGC CCC CGC TCC 3′ ZC872(Sequence ID Number 4) 5′ CCG GGG AGC GGG GGC TTG CCG GCC CTG GCA CTCATT TAC CTG AGG AAG AGA GAG CT 3′ ZC904 (Sequence ID Number 5) 5′ CATGGG CAC GTA ATC TAT AGA TTC ATC CTT GCT CAT ATC CAT GTA 3′ ZC905(Sequence ID Number 6) 5′ TCT TGC CAG GGC ACC TGG GAC ATC TGT TCC CACATC ACC GG 3′ ZC906 (Sequence ID Number 7) 5′ AAG CTG TCC TCT GCT TCAGCC AGA GGT CCT GGG CAG CC 3′ ZC1380 (Sequence ID Number 8) 5′ CAT GGTGGA ATT CCT GCT GAT 3′ ZC1447 (Sequence ID Number 9) 5′ TG GTT GTG CAGAGC TGA GGA AGA GAT GGA 3′ ZC1453 (Sequence ID Number 10) 5′ AAT TCA TTATGT TGT TGC AAG CCT TCT TGT TCC TGC TAG GTT TCG CTG TTA A 3′ ZC1454(Sequence ID Number 11) 5′ GAT CTT AAC AGC GAA ACC AGC TAG CAG GAA CAAGAA GGC CAA CAA CAT AAT G 3′ ZC1478 (Sequence ID Number 12) 5′ ATC GCGAGC ATG CAG ATC TGA 3′ ZC1479 (Sequence ID Number 13) 5′ AGC TTC AGA TCTGCA TGC TGC CGA T 3′ ZC1776 (Sequence ID Number 14) 5′ AGC TGA GCG CAAATG TTG TGT CGA GTG CCC ACC GTG CCC AGC TTA GAA TTC T 3′ ZC1777(Sequence ID Number 15) 5′ CTA GAG AAT TCT AAG CTG GGC ACG GTG GGC ACTCGA CAC AAC ATT TGC GCT C 3′ ZC1846 (Sequence ID Number 16) 5′ GAT CGGCCA CTG TCG GTG CGC TGC ACG CTG CGC     AAC GCT GTG GGC CAG GAC ACG CAGGAG GTC ATC    GTG GTG CCA CAC TCC TTG CCC TTT AAG CA 3′ ZC1847(Sequence ID Number 17) 5′ AGC TTG CTT AAA GGG CAA GGA GTG TGG CAC CAC   GAT GAC CTC CTG CGT GTC CTG GCC CAC AGC GTT    GCG CAG CGT GCA GCGCAC CGA CAG TGG CC 3′ ZC1886 (Sequence ID Number 18) 5′ CCA GTG CCA AGCTTG TCT AGA CTT ACC TTT AAA GGG CAA GGA G 3′ ZC1892 (Sequence ID Number19) 5′ AGC TTG AGC GT 3′ ZC1893 (Sequence ID Number 20) 5′ CTA GAC GCTCA 3′ ZC1894 (Sequence ID Number 21) 5′ AGC TTC CAG TTC TTC GGC CTC ATGTCA GTT CTT CGG CCT CAT GTG AT 3′ ZC1895 (Sequence ID Number 22) 5′ CTAGAT CAC ATG AGG CCG AAG AAC TGA CAT GAG GCC GAA GAA CTG GA 3′ ZC2181(Sequence ID Number 23) 5′ AAT TCG GAT CCA CCA TGG GCA CCA GCC ACC CGG   CGT TCC TGG TGT TAG GCT GCC TGC TGA CCG GCC 3′ ZC2182 (Sequence IDNumber 24) 5′ TGA GCC TGA TCC TGT GCC AAC TGA GCC TGC CAT    CGA TCC TGCCAA ACG AGA ACG AGA AGG TTG TGC    AGC TA 3′ ZC2183 (Sequence ID Number25) 5′ AAT TTA GCT GCA CAA CCT TCT CGT TCT CGT TTG    GCA GGA TCG ATGGCA GGC TCA GTT GGC ACA GGA    TCA 3′ ZC2184 (Sequence ID Number 26)5′ GGC TCA GGC CGG TCA GCA GGC AGC CTA ACA CCA    GGA ACG CCG GGT GGCTGG TGC CCA TGG TGG ATC     CG 3′ ZC2311 (Sequence ID Number 27) 5′ TGATCA CCA TGG CTC AAC TG 3′ ZC2351 (Sequence ID Number 28) 5′ CGA ATT CCAC 3′ ZC2352 (Sequence ID Number 29) 5′ CAT GGT GGA ATT CGA GCT 3′ ZC2392(Sequence ID Number 30) 5′ ACG TAA GCT TGT CTA GAC TTA CCT TCA GAA CGC   AGG GTG GG 3′

The 3′-end of the cDNA was not isolated in the first cloning and wassubsequently isolated by screening 6×10⁵ phage of the oligo d(T)-primedcDNA library with a 630 bp Sst I-Eco RI fragment derived from the 3′-endof clone RP41. One isolate, designated OT91, was further analyzed byrestriction enzyme mapping and DNA sequencing. This clone was found tocomprise the 3′-end of the receptor coding region and 1986 bp of 3′untranslated sequence.

Clones RP51, RP41 and OT91 were ligated together to construct afull-length cDNA encoding the entire PDGF β-receptor. RP41 was digestedwith Acc I and Bam HI to isolate the 2.12 kb fragment. RP51 was digestedwith Eco RI and Acc I to isolate the 982 bp fragment. The 2.12 kb RP41fragment and the 982 bp RP51 fragment were joined in a three-partligation with pUC13, which had been linearized by digestion with Eco RIand Bam HI. The resultant plasmid was designated 51/41. Plasmid 51/41was digested with Eco RI and Bam HI to isolate the 3 kb fragmentcomprising the partial PDGF receptor cDNA. OT91 was digested with Bam HIand Xba I to isolate the 1.4 kb fragment containing the 3′ portion ofthe PDGF receptor cDNA. The Eco RI-Bam HI 51/41 fragment, the Bam HI-XbaI OT91 fragment and the Eco RI-Xba I digested pUC13 were joined in athree-part ligation. The resultant plasmid was designated pR-RX1 (FIG.2).

B. Cloning the PDGF-α Receptor

A cDNA encoding to PDGF α-receptor was cloned as follows. RNA wasprepared by the method of Chirgwin et al. (Biochemistry 18: 5294, 1979)and twice purified on oligo d(T) cellulose to yield poly(A)+ RNA.Complementary DNA was prepared in λgt10 phage using a kit purchased fromInvitrogen (San Diego, Calif.). The resulting λ phage DNA was packagedwith a coat particle mixture from Stratagene Cloning Systems (La Jolla,Calif.), infected into E. coli strain C600 Hfl⁻ and titered.

Approximately 1.4×10⁶ phage recombinants were plated to produce plaquesfor screening. Nitrocellulose filter lifts were prepared according tostandard methods and were hybridized to a radiolabeled PDGF β-receptorDNA fragment (Gronwald et al., ibid.) comprising the 1.9 kb Fsp I-HindIII fragment that encodes the transmembrane and cytoplasmic domains ofthe PDGF β-receptor cDNA. Hybridization was performed for 36 hours at42° C. in a mixture containing 40% formamide, 5×SSCP (SSC containing 25mM phosphate buffer, pH 6.5), 200 μg/ml denatured salmon sperm DNA,3×Denhardt's, and 10% dextran sulfate. Following hybridization, thefilters were washed extensively at room temperature in 2×SSC, then for15 minutes at 47-48° C. Following an exposure to X-ray film, the filterswere treated to increasingly stringent wash conditions followed by filmrecording until a final wash with 0.1×SSC at 65° C. was reached. Filmanalysis showed that a “family” of plaques that hybridized at lower washstringency but not at the highest stringency. This “family” was selectedfor further analysis.

Two λ phage clones from the “family” obtained from the initial screeningwere subcloned into the Not I site of the pUCtype plasmid vectorpBluescript SK⁺ (obtained from Stratagene Cloning Systems, La Jolla,Calif.) and were analyzed by restriction mapping and sequence analysis.Restriction enzyme analysis of a phage clone, designated α1-1, revealeda restriction fragment pattern dissimilar from that of the PDGFβ-receptor cDNA with the exception of a common Bgl II-Bgl II band ofapproximately 160 bp. The PDGF β-receptor cDNA contains two similarlyspaced Bgl II sites within the region coding for the second tyrosinekinase domain.

Restriction analysis of a second plasmid subclone (designated α1-7)revealed an overlap of the 5′ approximately 1.2 kb of clone α1-1, and anadditional approximately 2.2 kb of sequence extending in the 5′direction. Sequence analysis revealed that the 3′ end of this cloneencodes the second tyrosine kinase domain, which contains regions ofnear sequence identity to the corresponding regions in the PDGFβ-receptor. The 5′ end of clone α1-7 contained non-receptor sequences.Two additional α-receptor clones were obtained by probing with α1-1sequences. Clone α1-1 was digested with Not I and Spe I, and a 230 bpfragment was recovered. Clone α1-1 was also digested with Bam HI and NotI, and a 550 bp fragment was recovered. A clone that hybridized to the230 bp probe was designated α5-1. This clone contained the 5′-mostcoding sequence for the PDGF α-receptor. Another clone, designated α6-3,hybridized to the 550 bp probe and was found to contain 3′ coding andnon-coding sequences, including the poly(A) tail.

Clone α1-1 was radiolabeled (³²p) and used to probe a northern blot(Thomas, Methods Enzymol. 100: 225-265, 1983) of the MG-63 poly(A)+ RNAused to prepare the cDNA library. A single band of approximately 6.6 kbwas observed. RNA prepared from receptor-positive cell lines includingthe human fibroblast SK4, WI-38 and 7573 cell lines; the mousefibroblast line DI 3T3; the U2-OS human osteosarcoma cell line andbaboon aortic smooth muscle cells, and RNA prepared fromreceptor-negative lines including A431 (an epithelial cell line) and VA13 (SV40-transformed WI-38 cells) were probed by northern format withthe α1-1 cDNA. In all cases, the amount of the 6.6 kb band detected inthese RNA correlated well with the relative levels of α-receptordetected on the respective cell surfaces. The 6.6 kb RNA was notdetected in RNA prepared from any tested cell line of hematopoieticorigin, in agreement with a lack of PDGF α-receptor protein detected onthese cell types.

Clones α1-1 and α1-7 were joined at a unique Pst I site in the regionencoding the transmembrane portion of the receptor. Clone α1-1 wasdigested with Xba I and Pst I and the receptor sequence fragment wasrecovered. Clone α1-7 was digested with Pst I and Bam HI and thereceptor fragment was recovered. The two fragments were ligated with XbaI+Bam HI-digested pIC19R (Marsh et al. Gene 32: 481-486, 1984) toconstruct plasmid pα17R (FIG. 12).

The remainder of the 5′-most α-receptor sequence was obtained from cloneα5-1 as an Sst I-Cla I fragment. This fragment was joined to the EcoRI-Sst I receptor fragment of pα17R and cloned into Eco RI+ClaI-digested pBluescript SK+ plasmid to construct plasmid pα17B (FIG. 12).FIG. 11 (Sequence ID Numbers 35 and 36) shows the nucleotide sequenceand deduced amino acid sequence of the cDNA present in pα17B.

Example 2 Construction of a SUC2 Signal Sequence-PDGF β-Receptor Fusion

To direct the PDGF β-receptor into the yeast secretory pathway, the PDGFβ-receptor cDNA was joined to a sequence encoding the Saccharomycescerevisiae SUC2 signal sequence. Oligonucleotides ZC1453 and ZC1454(Sequence ID Numbers 10 and 11; Table 1) were designed to form anadapter encoding the SUC2 secretory signal flanked by a 5′ Eco RIadhesive end and a 3′ Bgl II adhesive end. ZC1453 and ZC1454 wereannealed under conditions described by Maniatis et al. (ibid.). PlasmidpR-RX1 was digested with Bgl II and Sst II to isolate the 1.7 kbfragment comprising the PDGF β-receptor coding sequence from amino acids28 to 596. Plasmid pR-RX1 was also cut with Sst II and Hind III toisolate the 1.7 kb fragment comprising the coding sequence from aminoacids 597 through the translation termination codon and 124 bp of 3′untranslated DNA. The two 1.7 kb pR-RX1 fragments and the ZC1453/ZC1454adapter were joined with pUC19, which had been linearized by digestionwith Eco RI and Hind III. The resultant plasmid, comprising the SUC2signal sequence fused in-frame with the PDGF β-receptor cDNA, wasdesignated pBTL10 (FIG. 2).

Example 3 Construction of pCBS22

The BAR1 gene product, Barrier, is an exported protein that has beenshown to have three domains. When used in conjunction with a firstsignal sequence, the third domain of Barrier protein has been shown toaid in the secretion of proteins into the medium (MacKay et al., U.S.patent application Ser. No. 07/104,316), now abandoned.

The portion of the BAR1 gene encoding the third domain of Barrier wasjoined to a sequence encoding the C-terminal portion of substance P(subP; Munro and Pelham, EMBO J. 3: 3087-3093, 1984). The presence ofthe substance P amino acids on the terminus of the fusion proteinallowed the protein to be detected using commercially availableanti-substance P antibodies. Plasmid pZV9 (deposited as a transformantin E. coli strain RR1, ATCC accession no. 53283), comprising the entireBAR1 coding region and its associated flanking regions, was cut with SalI and Bam HI to isolate the 1.3 kb BAR1 fragment. This fragment wassubcloned into pUC13, which had been cut with Sal I and Bam HI, togenerate the plasmid designated pZV17. Plasmid pZV17 was digested withEco RI to remove the 3′-most 0.5 kb of the BAR1 coding region. Thevector-BAR1 fragment was religated to create the plasmid designatedpJH66 (FIG. 3). Plasmid pJH66 was linearized with Eco RI and blunt-endedwith DNA polymerase I (Klenow fragment). Kinased Bam HI linkers (5° CCGGAT CCG G 3′) were added and excess linkers were removed by digestionwith Bam HI before religation. The resultant plasmid was designated pSW8(FIG. 3).

Plasmid pSW81, comprising the TPI1 promoter, the BAR1 coding regionfused to the coding region of the C-terminal portion of substance P(Munro and Pelham, EMBO J. 3: 3087-3093, 1984) and the TPI1 terminator,was derived from pSW8. Plasmid pSW8 was cut with Sal I and Bam HI toisolate the 824 bp fragment encoding amino acids 252 through 526 ofBAR1. Plasmid pPM2, containing the synthetic oligonucleotide sequenceencoding the dimer form of the C-terminal portion of substance P (subP)in M13mp8, was obtained from Hugh Pelham (MRC Laboratory of MolecularBiology, Cambridge, England). Plasmid pPM2 was linearized by digestionwith Bam HI and Sal I and ligated with the 824 bp BAR1 fragment frompSW8. The resultant plasmid, pSW14, was digested with Sal I and Sma I toisolate the 871 bp BAR1-substance P fragment. Plasmid pSW16, comprisinga fragment of BAR1 encoding amino acids 1 through 250, was cut with XbaI and Sal I to isolate the 767 bp BAR1 fragment. This fragment wasligated with the 871 bp BAR1-substance P fragment in a three-partligation with pUC18 cut with Xba I and Sma I. The resultant plasmid,designated pSW15, was digested with Xba I and Sma I to isolate the 1.64kb BAR1-substance P fragment. The ADH1 promoter was obtained frompRL029. Plasmid pRL029, comprising the ADH1 promoter and the BAR1 5′region encoding amino acids 1 to 33 in pUC18, was digested with Sph Iand Xba I to isolate the 0.42 kb ADH1 promoter fragment. The TPI1terminator (Alber and Kawasaki, ibid.) was provided as a linearizedfragment containing the TPI1 terminator and pUC18 with a Klenow-filledXba I end and an Sph I end. This fragment was ligated with the 0.42 kbADH1 promoter fragment and the 1.64 kb BAR1-substance P fragment in athree-part ligation to produce plasmid pSW22.

The ADH1 promoter and the coding region of BAR1, from the translationinitiation ATG through the Eco RV site present in pSW22, were removed bydigestion with Hind III and Eco RV. The 3.9 kb vector fragment,comprising the 401 bp between the Eco RV and the Eco RI sites of theBAR1 gene fused to subP and the TPI1 terminator, was isolated by gelelectrophoresis. Oligonucleotide ZC1478 (Sequence ID Number 12; Table 1)was kinased and annealed with oligonucleotide ZC1479 (Sequence ID Number13; Table 1) using conditions described by Maniatis et al. (ibid.). Theannealed oligonucleotides formed an adapter comprising a Hind IIIadhesive end and a polylinker encoding Bgl II, Sph I, Nru I and Eco RVrestriction sites. The ZC1479/ZC1478 adapter was ligated with thegel-purified pSW22 fragment. The resultant plasmid was designated pCBS22(FIG. 3).

Example 4 Construction of pBTL13

In order to enhance the secretion of the PDGF β-receptor and tofacilitate the identification of the secreted protein, a sequenceencoding the third domain of BAR1 fused to the C-terminal amino acids ofsubstance P was fused in frame with the 5′1240 bp of the PDGFβ-receptor. Plasmid pBTL10 (Example 2) was digested with Sph I and Sst Ito isolate the 4 kb fragment comprising the SUC2 signal sequence, aportion of the PDGF β-receptor cDNA and the pUC19 vector sequences.Plasmid pCBS22 was digested with Sph I and Sst I to isolate the 1.2 kbfragment comprising the BAR1-subP fusion and the TPI1 terminator. Thesetwo fragments were ligated, and the resultant plasmid was designatedpBTL13 (FIG. 4).

Example 5 Construction of an Expression Vector Encoding the Entire PDGFβ-Receptor

The entire PDGF β-receptor was directed into the secretory pathway byfusing a SUC2 signal sequence to the 5′ end of the PDGF β-receptorcoding sequence. This fusion was placed behind the TPI1 promoter andinserted into the vector YEp13 for expression in yeast.

The TPI1 promoter was obtained from plasmid pTPIC10 (Alber and Kawasaki,J. Mol. Appl. Genet. 1: 410-434, 1982), and plasmid pFATPOT (Kawasakiand Bell, EP 171,142; ATCC 20699). Plasmid pTPIC10 was cut at the uniqueKpn I site, the TPI1 coding region was removed with Bal-31 exonuclease,and an Eco RI linker (sequence: GGA ATT CC) was added to the 3′ end ofthe promoter. Digestion with Bgl II and Eco RI yielded a TPI1 promoterfragment having Bgl II and Eco RI sticky ends. This fragment was thenjoined to plasmid YRp7′ (Stinchcomb et al., Nature 282: 39-43, 1979)that had been cut with Bgl II and Eco RI (partial). The resultingplasmid, TE32, was cleaved with Eco RI (partial) and Bam HI to remove aportion of the tetracycline resistance gene. The linearized plasmid wasthen recircularized by the addition of an Eco RI-Bam HI linker toproduce plasmid TEA32. Plasmid TEA32 was digested with Bgl II and EcoRI, and the 900 bp partial TPI1 promoter fragment was gel-purified.Plasmid pIC19H (Marsh et al., Gene 32:481-486, 1984) was cut with Bgl IIand Eco RI and the vector fragment was gel purified. The TPI1 promoterfragment was then ligated to the linearized pIC19H and the mixture wasused to transform E. coli RR1. Plasmid DNA was prepared and screened forthe presence of at ˜900 bp Bgl II-Eco RI fragment. A correct plasmid wasselected and designated pICTPIP.

The TPI1 promoter was then subcloned to place convenient restrictionsites at its ends. Plasmid pIC7 (Marsh et al., ibid.) was digested withEco RI, the fragment ends were blunted with DNA polymerase I (Klenowfragment), and the linear DNA was recircularized using T4 DNA ligase.The resulting plasmid was used to transform E. coli RR1. Plasmid DNA wasprepared from the transformants and was screened for the loss of the EcoRI site. A plasmid having the correct restriction pattern was designatedpIC7RI*. Plasmid pIC7RI* was digested with Hind III and Nar I, and the2500 bp fragment was gel-purified. The partial TPI1 promoter fragment(ca. 900 bp) was removed from pICTPIP using Nar I and Sph I and wasgel-purified. The remainder of the TPI1 promoter was obtained fromplasmid pFATPOT by digesting the plasmid with Sph I and Hind III, and a1750 bp fragment, which included a portion of the TPI1 promoter fragmentfrom pICTPIP, and the fragment from pFATPOT were then combined in atriple ligation to produce pMVR1 (FIG. 2).

The TPI1 promoter was then joined to the SUC2-PDGF β-receptor fusion.Plasmid pBTL10 (Example 2) was digested with Eco RI and Hind III toisolate the 3.4 kb fragment comprising the SUC2 signal sequence and theentire PDGF β-receptor coding region. Plasmid pMVR1 was digested withBgl II and Eco RI to isolate the 0.9 kb TPI1 promoter fragment. The TPI1promoter fragment and the fragment derived from pBTL10were joined withYEp13, which had been linearized by digestion with Bam HI and Hind III,in a three-part ligation. The resultant plasmid was designated pBTL12(FIG. 2).

Example 6 Construction of an Expression Vector Encoding the 5′Extracellular Portion of the PDGF β-Receptor

The extracellular portion of the PDGF β-receptor was directed into thesecretory pathway by fusing the coding sequence to the SUC2 signalsequence. This fusion was placed in an expression vector behind the TPI1promoter. Plasmid pBTL10 (Example 2) was digested with Eco RI and Sph Ito isolate the approximately 1.3 kb fragment comprising the SUC2 signalsequence and the PDGF β-receptor extracellular domain coding sequence.Plasmid pMVR1 (Example 5) was digested with Bgl II and Eco RI to isolatethe 0.9 kb TPI1 promoter fragment. The TPI1 promoter fragment was joinedwith the fragment derived from pBTL10 and YEp13, which had beenlinearized by digestion with Bam HI and Sph I, in a three-part ligation.The resultant plasmid was designated pBTL11 (FIG. 2).

Example 7 Construction of Yeast Expression Vectors pBTL14 and pBTL15,and The Expression of PDGF β-Receptor-BAR1-subP Fusions

A. Construction of pBTL14

The SUC2-PDGFβ-R fusion was joined with the third domain of BAR1 toenhance the secretion of the receptor, and the expression unit wascloned into a derivative of YEp13 termed pJH50. YEp13 was modified todestroy the Sal I site near the LEU2 gene. This was achieved bypartially digesting YEp13 with Sal I followed by a complete digestionwith Xho I. The 2.0 kb Xho I-Sal I fragment comprising the LEU2 gene andthe 8.0 kb linear YEp13 vector fragment were isolated and ligatedtogether. The ligation mixture was transformed into E. coli strain RR1.DNA was prepared from the transformants and was analyzed by digestionwith Sal I and Xho I. A clone was isolated which showed a single Sal Isite and an inactive Xho I site indicating that the LEU2 fragment hadinserted in the opposite orientation relative to the parent plasmidYEp13. The plasmid was designated pJH50.

Referring to FIG. 4, plasmid pBTL12 (Example 5) was digested with Sal Iand Pst I to isolate the 2.15 kb fragment comprising 270 bp of YEp13vector sequence, the TPI1 promoter, the SUC2 signal sequence, and 927 bpof PDGF β-receptor cDNA. Plasmid pBTL13 (Example 4) was digested withPst I and Hind III to isolate the 1.48 kb fragment comprising 313 bp ofPDGF β-receptor cDNA, the BAR1-subP fusion and the TPI1 terminator. Thefragments derived from pBTL12 and pBTL13 were joined with pJH50, whichhad been linearized by digestion with Hind III and Sal I, in athree-part ligation. The resultant plasmid was designated pBTL14.

B. Construction of pBTL15

Referring to FIG. 5, a yeast expression vector was constructedcomprising the TPI1 promoter, the SUC2 signal sequence, 1.45 kb of PDGFβ-receptor cDNA sequence fused to the BAR1-subP fusion and the TPI1terminator. Plasmid pBTL12 (Example 5) was digested with Sal I and Fsp Ito isolate the 2.7 kb fragment comprising the TPI1 promoter, the SUC2signal sequence, the PDGFβ-R coding sequence, and 270 bp of YEp13 vectorsequence. Plasmid pBTL13 (Example 4) was digested with Nru I and HindIII to isolate the 1.4 kb fragment comprising the BAR1-subP fusion, theTPI1 terminator and 209 bp of 3′ PDGF β-receptor cDNA sequence. Thefragments derived from pBTL12 and pBTL13 were joined in a three-partligation with pJH50, which had been linearized by digestion with HindIII and Sal I. The resultant plasmid was designated pBTL15.

C. Expression of PDGFβ-R-subP fusions from pBTL14 and pBTL15

Yeast expression vectors pBTL14 and pBTL15 were transformed intoSaccharomyces cerevisiae strains ZY100 (MATa leu2-3,112 ade2-101 suc2-Δ9gal2 pep4::TPI1prom-CAT) and ZY400 (MATa leu2-3,112 ade2-101 suc2-Δ9gal2 pep4::TPI1prom-CAT Δmnn9::URA3). Transformations were carried outusing the method essentially described by Beggs (ibid.). Transformantswere selected for their ability to grow on -LEUDS (Table 2).

Table 2

TABLE 2 Media Recipes -LeuThrTrp Amino Acid Mixture 4 g adenine 3 gL-arginine 5 g L-aspartic acid 2 g L-histidine free base 6 gL-isoleucine 4 g L-lysine-mono hydrochloride 2 g L-methionine 6 gL-phenylalanine 5 g L-serine 5 g L-tyrosine 4 g uracil 6 g L-valine

Mix all the ingredients and grind with a mortar and pestle until themixture is finely ground.

-LEUDS 20 g glucose 6.7 g Yeast Nitrogen Base without amino acids (DIFCOLaboratories Detroit, MI) 0.6 g -LeuThrTrp Amino Acid Mixture 182.2 gsorbitol 18 g Agar

Mix all the ingredients in distilled water. Add distilled water to afinal volume of 1 liter. Autoclave 15 minutes. After autoclaving add 150mg L-threonine and 40 mg L-tryptophan. Pour plates and allow tosolidify.

-LEUDS + sodium succinate, pH 6.5 20 g Yeast Nitrogen Base without aminoacids 0.6 g -LeuTrpThr Amino Acid Mixture 182.2 g sorbitol 11.8 gsuccinic acid

Mix all ingredients in distilled water to a final volume of 1 liter.Adjust the pH of the solution to pH 6.5. Autoclave 15 minutes. Afterautoclaving add 150 mg L-threonine and 40 mg L-tryptophan.

Fermentation Medium 7 g/l yeast nitrogen base without amino acids orammonium sulfate (DIFCO Laboratories) 0.6 g/l ammonium sulfate 0.5Msorbitol 0.39 g/l adenine sulfate 0.01% polypropylene glycol

Mix all ingredients in distilled water. Autoclave 15 minutes. Add 80 ml50% glucose for each liter of medium.

Super Synthetic-LEUD, pH 6.5 (liquid or solid medium) 6.7 g YeastNitrogen Base without amino acids or ammonium sulfate (DIFCO) 6 gammonium sulfate 160 g adenine 0.6 g -LeuThrTrp Amino Acid Mixture 20 gglucose 11.8 g succinic acid

Mix all ingredients and add distilled water to a final volume of 800 ml.Adjust the pH of the solution to pH 6.4. Autoclave 15 minutes. For solidmedium, add 18 g agar before autoclaving, autoclave and pour plates.

Super Synthetic-LEUDS, pH 6.4 (Liquid or Solid Medium)

Use the same recipe as Super Synthetic -LEUD, pH 6.4, but add 182.2 gsorbitol before autoclaving.

YEPD 20 g glucose 20 g Bacto Peptone (DIFCO Laboratories) 10 g BactoYeast Extract (DIFCO Laboratories) 18 g agar 4 ml adenine 1% 8 ml 1%L-leucine

Mix all ingredients in distilled water, and bring to a final volume of 1liter. Autoclave 25 minutes and pour plates.

The transformants were assayed for binding to an anti-PDGF β-receptormonoclonal antibody (PR7212) or an anti-substance P antibody by proteinblot assay. ZY100[pBTL14] and ZY100[pBTL15] transformants were grownovernight at 30° C. in 5 ml Super Synthetic -LEUD, pH 6.4 (Table 2).ZY400[pBTL14] and ZY400[pBTL15] transformants were grown overnight at30° C. in 5 ml Super Synthetic-LEUDS, pH 6.4 (Table 2). The cultureswere pelleted by centrifugation and the supernatants were assayed forthe presence of secreted PDGF β-receptor analogs by protein blot assayusing methods described in Example 18. Results of assays using PR7212are shown in Table 3.

TABLE 3 Results of a protein blot probed with PR7212 Transformant:ZY100[pBTL14] + ZY400[pBTL14] ++ ZY100[pBTL15] + ZY400[pBTL15] +

Example 8 Construction of a SUC2-PDGFβ-R Fusion Comprising the CompletePDGFβ-R Extracellular Domain

A. Construction of pBTL22

The PDGFβ-R coding sequence present in pBTL10 was modified to delete thecoding region 3′ to the extracellular PDGFβ-R domain. As shown in FIG.6, plasmid pBTL10 was digested with Sph I and Bam HI and with Sph I andSst II to isolate the 4.77 kb fragment and the 466 bp fragment,respectively. The 466 bp fragment was then digested with Sau 3A toisolate the 0.17 kb fragment. The 0.17 kb fragment and the 4.77 kb werejoined by ligation. The resultant plasmid was designated pBTL21.

Plasmid pBTL21, containing a Bam HI site that was regenerated by theligation of the Bam HI and Sau 3A sites, was digested with Hind III andBam HI to isolate the 4.2 kb fragment. Synthetic oligonucleotides ZC1846(Sequence ID Number 16; Table 1) and ZC1847 (Sequence ID Number 17;Table 1) were designed to form an adapter encoding the PDGFβ-R from theSau 3A site after bp 1856 (FIGS. 1A-1G; (Sequence ID Number 1)) to theend of the extracellular domain at 1958 bp (FIGS. 1A-1G; Sequence IDNumber 1), having a 5′ Bam HI adhesive end that destroys the Bam HI siteand a 3′ Hind III adhesive end. Oligonucleotides ZC1846 and ZC1847 wereannealed under conditions described by Maniatis et. al. (ibid.). The 4.2kb pBTL21 fragment and the ZC1846/ZC1847 adapter were joined byligation. The resultant plasmid, designated pBTL22, comprises the SUC2signal sequence fused in proper reading frame to the extracellulardomain of PDGFβ-R in the vector pUC19 (FIG. 6).

B. Construction of pBTL28

An in-frame translation stop codon was inserted immediately after thecoding region of the PDGFβ-R in pBTL22 using oligonucleotides ZC1892(Sequence ID Number 19; Table 1) and ZC1893 (Sequence ID Number 20;Table 1). These oligonucleotides were designed to form an adapterencoding a stop codon in-frame with the PDGFβ-R coding sequence frompBTL22 flanked by a 5′ Hind III adhesive end and a 3′ Xba I adhesiveend. Plasmid pBTL22 was digested with Eco RI and Hind III to isolate the1.6 kb SUC2-PDGFβ-R fragment. Plasmid pMVR1 was digested with Eco RI andXba I to isolate the 3.68 kb fragment comprising the TPI1 promoter,pIC7RI* vector sequences and the TPI1 terminator. OligonucleotidesZC1892 and ZC1893 were annealed to form a Hind III-Xba I adapter. The1.6 kb SUC2-PDGFβ-R fragment, the 3.86 kb pMVR1 fragment and theZC1892/ZC1893 adapter were joined in a three-part ligation. Theresultant plasmid was designated pBTL27.

The expression unit present in pBTL27 was inserted into the yeastexpression vector pJH50 by first digesting pJH50 with Bam HI and Sal Ito isolate the 10.3 kb vector fragment. Plasmid pBTL27 was digested withBgl II and Eco RI and with Xho I and Eco RI to isolate the 0.9 kb TPI1promoter fragment and the 1.65 kb fragment, respectively. The 10.3 kbpJH50 vector fragment, the 0.9 kb TPI1 promoter fragment and 1.65 kbfragment were joined in a three-part ligation. The resultant plasmid wasdesignated pBTL28.

C. Construction of Plasmid pBTL30

The PDGFβ-R coding sequence present in plasmid pBTL22 was modified toencode the twelve C-terminal amino acids of substance P and an in-framestop codon. Plasmid pBTL22 was digested with Eco RI and Hind III toisolate the 1.6 kb SUC2-PDGFβ-R fragment. Plasmid pMVR1 was digestedwith Eco RI and Xba I to isolate the 3.68 kb fragment comprising theTPI1 promoter, pIC7RI* and the TPI1 terminator. Syntheticoligonucleotides ZC1894 (Sequence ID Number 21; Table 1 and ZC1895(Sequence ID Number 22; Table 1) were annealed to form an adaptercontaining the codons for the twelve C-terminal amino acids of substanceP followed by an in-frame stop codon and flanked on the 5′ end with aHind III adhesive end and on the 3′ end with an Xba I adhesive end. TheZC1894/ZC1895 adapter, the 1.6 kb, SUC2-PDGFβ-R fragment and the pMVR1fragment were joined in a three-part ligation. The resultant plasmid,designated pBTL29, was digested with Eco RI and Xho I to isolate the 1.69 kb SUC2-PDGFβ-R-subP-TPI1 terminator fragment. Plasmid pBTL27 wasdigested with Bgl II and Eco RI to isolate the 0.9 kb TPI1 promoterfragment. Plasmid pJH50 was digested with Bam HI and Sal I to isolatethe 10.3 kb vector fragment. The 1.69 kb pBTL29 fragment, the 0.9 kbTPI1 promoter fragment and the 10.3 kb vector fragment were joined in athree-part ligation. The resulting plasmid was designated pBTL30.

Example 9 Construction and Expression of a SUC2-PDGFβ-R-IgG HingeExpression Vector

An expression unit comprising the TPI1 promoter, the SUC2 signalsequence, the PDGFβ-R extracellular domain, an immunoglobulin hingeregion and the TPI1 terminator was constructed. Plasmid pBTL22 wasdigested with Eco RI and Hind III to isolate the 1.56 kb fragment.Plasmid pMVR1 was digested with Eco RI and Xba I to isolate the 3.7 kbfragment, comprising the TPI1 promoter, pIC7RI* vector sequences and theTPI1 terminator. oligonucleotides ZC1776 (Sequence ID Number 14;Table 1) and ZC1777 (Sequence ID Number 15; Table 1) were designed toform, when annealed, an adapter encoding an immunoglobulin hinge regionwith a 5′ Hind III adhesive end and a 3′ Xba I adhesive end.Oligonucleotides ZC1776 and ZC1777 were annealed under conditionsdescribed by Maniatis et al. (ibid.). The 1.5kb pBTL22 fragment, the 3.7kb fragment and the ZC1776/ZC1777 adapter were joined in a three-partligation, resulting in plasmid pBTL24.

The expression unit of pBTL24, comprising the TPI1 promoter, SUC2 signalsequence, PDGFβ-R extracellular domain sequence, hinge region sequence,and TPI1 terminator, was inserted into pJH50. Plasmid pBTL24 wasdigested with Xho I and Hind III to isolate the 2.4 kb expression unit.Plasmid pJH50 was digested with Hind III and Sal I to isolate the 9.95kb fragment. The 2.4 kb pBTL24 fragment and 9.95 kb pJH50 vectorfragment were joined by ligation. The resultant plasmid was designatedpBTL25.

Plasmid pBTL25 was transformed into Saccharomyces cerevisiae strainZY400 using the method essentially described by Beggs (ibid.).Transformants were selected for their ability to grow on -LEUDS (Table2). The transformants were tested for their ability to bind theanti-PDGFβ-R monoclonal antibody PR7212 using the colony assay methoddescribed in Example 18. Plasmid pBTL25 transformants were patched ontonitrocellulose filters that had been wetted and supported by YEPD solidmedium. Antibody PR7212 was found to bind to the PDGFβ-R-IgG hingefusion secreted by ZY400[pBTL25] transformants.

Example 10 Construction and Expression of a SUC2 signal sequence-PDGFβ-RExtracellular Domain-SUC2 Fusion

As shown in FIG. 6, an expression unit comprising the TPI1 promoter,SUC2 signal sequence, PDGFβ-R extracellular domain sequence, and SUC2coding sequence was constructed as follows. Plasmid pBTL22 was digestedwith Eco RI and Hind III to isolate the 1.6 kb SUC2-PDGFβ-R fragment.Plasmid pMVR1 was digested with Bgl II and Eco RI to isolate the 0.9 kbTPI1 promoter fragment. The SUC2 coding region was obtained from pJH40.Plasmid pJH40 was constructed by inserting the 2.0 kb Hind III-Hind IIISUC2 fragment from pRB58 (Carlson et al., Cell 28:145-154, 1982) intothe Hind III site of pUC19 followed by the destruction of the Hind IIIsite 3′ to the coding region. Plasmid pJH40 was digested with Hind IIIand Sal I to isolate the 2.0 kb SUC2 coding sequence. Plasmid pJH50 wasdigested with Sal I and Bam HI to isolate the 10.3 kb vector fragment.The 0.9 kb Bgl II-Eco RI TPI1 promoter fragment, the 1.6 kb Eco RI-HindIII SUC2-PDGFβ-R, the 2.0 kb Hind III-Sal I SUC2 fragment and the 10.3kb Bam HI-Sal I vector fragment were joined in a four-part ligation. Theresultant plasmid was designated pBTL26 (FIG. 6).

Plasmid pBTL26 was transformed into Saccharomyces cerevisiae strainZY400 using the method essentially described by Beggs (ibid.).Transformants were selected for their ability to grow on -LEUDS (Table2). ZY400 transformants (ZY400[pBTL26]) were assayed by protein blot(Example 18), colony blot (Example 18) and competition assay.

Protein blot assays were carried out on ZY400[pBTL26] and ZY400[pJH50](control) transformants that had been grown in flasks. Two hundred-fiftymicroliters of a 5 ml overnight cultures of ZY400[pBTL26] and ZY400[pJH50] in -LEUDS+sodium succinate, pH 6.5 (Table 2) were inoculatedinto 50 ml of -LEUDS+sodium succinate, pH 6.5. The cultures wereincubated for 35 hours in an airbath shaker at 30° C. The culturesupernatants were harvested by centrifugation. The culture supernatantswere assayed as described in Example 18 and were found to bind PR7212antibody.

Colony assays were carried out on ZY400[pBTL26] transformants.ZY400[pBTL26] transformants were patched onto wetted nitrocellulosefilters that were supported on a YEPD plate. The colony assay carriedout as described in Example 8.A. showed that ZY400[pBTL26] antibodiesbound PR7212 antibodies.

Competition binding assays were carried out on ZY400[pBTL26] andZY400[pJH50] transformants. The transformants were grown in two litersof fermentation medium (Table 2) in a New Brunswick Bioflo2 fermentor(New Brunswick, Philadelphia, Pa.) with continuous pH control at pH 6.4.The cultures were adjusted to pH 7.5 immediately prior to harvesting.Culture supernatants were concentrated in an Amicon concentrator(Amicon, San Francisco, Calif.) using an Amicon 10⁴ mw spiral filtercartridge. The concentrated supernatants were further concentrated usingAmicon Centriprep 10's. Fifteen milliliters of the concentratedsupernatant samples were added to the Centripreps, and the Centriprepswere spun in a Beckman GRP centrifuge (Beckman Instruments Inc.,Carlsbad, Calif.) at setting 5 for a total of 60 minutes. Theconcentrates were removed from the Centripreps and were assayed in thecompetition assay.

The competition binding assay measured the amount of ¹²⁵I-PDGF left tobind to fetal foreskin fibroblast cells after preincubation with theconcentrate containing the PDGFβ-R-SUC2 fusion protein. PDGF-AA andPDGF-AB were iodinated using the Iodopead method (Pierce Chemical).PDGF-BB_(Tyr) was iodinated and purified as described in Example 18.F.The concentrate was serially diluted in binding medium (Table 4). Thedilutions were mixed with 0.5 ng of iodinated PDGF-AA, PDGF-BB_(Tyr) orPDGF-AB, and the mixtures were incubated for two hours at roomtemperature. Three hundred micrograms of unlabeled PDGF-BB was added toeach sample mixture. The sample mixtures were added to 24-well platescontaining confluent fetal foreskin fibroblast cells (AG1523, availablefrom the Human Genetic Mutant Cell Repository, Camden, N.J.). The cellswere incubated with the mixture for four hours at 4° C. The supernatantswere aspirated from the wells, and the wells were rinsed three timeswith phosphate buffered saline that was held at 4° C. (PBS; Sigma, St.Louis, Mo.). Five hundred microliters of PBS+1% NP-40 was added to eachwell, and the plates were shaken on a platform shaker for five minutes.The cells were harvested and the amount of iodinated PDGF wasdetermined. The results of the competition binding assay showed that thePDGFβ-R-SUC2 fusion protein was able to competitively bind all threeisoforms of PDGF.

The PDGFβ-R produced from ZY400 [pBTL26] transformants was tested forcross reactivity to fibroblast growth factor (FGF) and transforminggrowth factor-β (TGF-β) using the competition assay essentiallydescribed above. Supernatant concentrates from ZY400[pBTL26] andZY400[JH50] (control) transformants were serially diluted in bindingmedium (Table 4). The dilutions were mixed with 7.9 ng of iodinated FGFor 14 ng of iodinated TGF-β, and the mixtures were incubated for twohours at room temperature. Fourteen micrograms of unlabeled FGF wasadded to each mixture containing labeled FGF, and 7 μg of unlabeledTGF-β was added to each mixture containing labeled TGF-β. The samplemixtures were added to 24-well plates containing confluent human dermalfibroblast cells. (Human dermal fibroblast cells express both FGFreceptors and TGFβ receptors.) The cells were incubated with themixtures for four hours at 4° C. Five hundred microliters of PBS+1%NP-40 was added to each well, and the plates were shaken on a platformshaker for five minutes. The cells were harvested and the amount ofiodinated FGF or TGF-β bound to the cells was determined. The results ofthese assays showed that the PDGFβ-R-SUC2 fusion protein did not crossreact with FGF or TGF-β.

TABLE 4 Reagent Recipes Binding Medium 500 ml Ham's F-12 medium 12 ml 1MHEPES, pH 7.4 5 ml 100x PSN (Penicillin/Streptomycin/Neomycin, Gibco) 1g rabbit serum albumin Western Transfer Buffer 25 mM Tris, pH 8.3 19 mMglycine, pH 8.3 20% methanol Western Buffer A 50 ml 1M Tris, pH 7.4 20ml 0.25 mM EDTA, pH 7.0 5 ml 10% NP-40 37.5 ml 4M NaCl 2.5 g gelatin

The Tris, EDTA, NP-40 and NaCl were diluted to a final volume of oneliter with distilled water. The gelatin was added to 300 ml of thissolution and the solution was heated in a microwave until the gelatinwas in solution. The gelatin solution was added back to the remainder ofthe first solution and stirred at 4° C. until cool. The buffer wasstored at 4° C.

Western Buffer B 50 ml 1M Tris, pH 7.4 20 ml 0.25M EDTA, pH 7.0 5 ml 10%NP-40 58.4 g NaCl 2.5 g gelatin 4 g N-lauroyl sarcosine

The Tris, EDTA, NP-40, and the NaCl were mixed and diluted to a finalvolume of one liter. The gelatin was added to 300 ml of this solutionand heated in a microwave until the gelatin was in solution. The gelatinsolution was added back to the original solution and the N-lauroylsarcosine was added. The final mixture was stirred at 4° C. until thesolids were completely dissolved. This buffer was stored at 4° C.

2x Loading Buffer 36 ml 0.5M Tris-HCl, pH 6.8 16 ml glycerol 16 ml 20%SDS 4 ml 0.5% Bromphenol Blue in 0.5M Tris-HCl, pH 6.8

Mix all ingredients. Immediately before use, add 100 μlβ-mercaptoethanol to each 900 μl dye mix

Example 11 Construction and Expression of PDGF Receptor Analogs From BHKcells

A. Construction of pBTL114 and pBTL115

The portions of the PDGF β-receptor extracellular domain present inpBTL14 and pBTL15 were placed in a mammalian expression vector. PlasmidspBTL14 and pBTL15 were digested with Eco RI to isolate the 1695 bp and1905 bp SUC2 signal-PDGFβ-R-BAR1 fragments. The 1695 bp fragment and the1905 bp fragment were each ligated to Zem229R that had been linearizedby digestion with Eco RI.

The vector Zem229R was constructed as shown in FIG. 10 from Zem229.Plasmid Zem229 is a pUC18-based expression vector containing a uniqueBam HI site for insertion of cloned DNA between the mousemetallothionein-1 promoter and SV40 transcription terminator and anexpression unit containing the SV40 early promoter, mouse dihydrofolatereductase gene, and SV40 transcription terminator. Zem229 was modifiedto delete the Eco RI sites flanking the Bam HI cloning site and toreplace the Bam HI site with a single Eco RI cloning site. The plasmidwas partially digested with Eco RI, treated with DNA polymerase I(Klenow fragment) and dNTPs, and religated. Digestion of the plasmidwith Bam HI followed by ligation of the linearized plasmid with a BamHI-Eco I adapter resulted in a unique Eco RI cloning site. The resultantplasmid was designated Zem229R.

The ligation mixtures were transformed into E. coli strain RR1. PlasmidDNA was prepared and the plasmids were subjected to restriction enzymeanalysis. A plasmid having the 1695 bp pBTL14 fragment inserted intoZem229R in the correct orientation was designated pBTL114 (FIG. 9). Aplasmid having the 1905 bp pBTL15 fragment inserted into Zem229R in thecorrect orientation was designated pBTL115 (FIG. 9).

B. Expression of Secreted PDGF β-receptor Analogs in tk⁻ ts13 BHK Cells

Plasmids pBTL114 and pBTL115 were each transfected into tk⁻ts13 cellsusing calcium phosphate precipitation (essentially as described byGraham and van der Eb, J. Gen. Virol. 36: 59-72, 1977). The transfectedcells were grown in Dulbecco's modified Eagle's medium (DMEM) containing10% fetal calf serum, 1×PSN antibiotic mix (Gibco 600-5640), 2.0 mML-glutamine. The cells were selected in 250 nM methotrexate (MTX) for 14days, and the resulting colonies were screened by the immunofilter assay(McCracken and Brown, Biotechniques, 82-87, March/April 1984). Plateswere rinsed with PBS or No Serum medium (DMEM plus 1×PSN antibioticmix). Teflon® mesh (Spectrum Medical Industries, Los Angeles, Calif.)was then placed over the cells. Nitrocellulose filters were wetted withPBS or No Serum medium, as appropriate, and placed over the mesh. Aftersix hours incubation at 37° C., filters were removed and placed inWestern buffer A (Table 4) overnight at room temperature. The filterswere developed using the antibody PR7212 and the procedure described inExample 8. The filters showed that conditioned media frompBTL114-transfected and pBTL115-transfected BHK cells bound the PR7212antibody indicating the presence of biologically active secretedPDGFβ-R.

Example 12

Expression of PDGF β-Receptor Analogs in Cultured Mouse Myeloma Cells

A. Construction of pICμPRE8

The immunoglobulin μ heavy chain promoter and enhancer were subclonedinto pIC19H to provide a unique Hind III site 3′ to the promoter.Plasmid pμ (Grosschedl and Baltimore, Cell 41: 885-897, 1985) wasdigested with Sal I and Eco RI to isolate the 3.1 kb fragment comprisingthe μ promoter. Plasmid pIC19H was linearized by digestion with Eco RIand Xho I. The μ promoter fragment and the linearized pIC19H vectorfragment were joined by ligation. The resultant plasmid, designatedpICμ3, was digested with Ava II to isolate the 700 bp μ promoterfragment. The 700 bp fragment was blunt-ended by treatment with DNApolymerase I (Klenow fragment) and deoxynucleotide triphosphates.Plasmid pIC19H was linearized by digestion with Xho I, and the adhesiveends were filled in by treatment with DNA polymerase I (Klenow fragment)and deoxynucleotide triphosphates. The blunt-ended Ava II fragment wasligated with the blunt-ended, linearized pIC19H, and the ligationmixture was transformed into E. coli JM83. Plasmid DNA was prepared fromthe transformants and was analyzed by restriction digest. A plasmid witha Bgl II site 5′ to the promoter was designated pICμPR1(−). PlasmidpICμPR1(−) was digested with Hind III and Bgl II to isolate the 700 bp μpromoter fragment. Plasmid pIC19R was linearized by digestion with HindIII and Bam HI. The 700 bp promoter fragment was joined with thelinearized pIC19R by ligation. The resultant plasmid, designatedpICμPR7, comprised the μ promoter with an unique Sma I site 5′ to thepromoter and a unique Hind III site 3′ to the promoter.

The immunoglobulin heavy chain μ enhancer (Gillies et al., Cell 33:717-728, 1983) was inserted into the unique Sma I site to generateplasmid pICμPRE8. Plasmid pJ4 (obtained from F. Blattner, Univ.Wisconsin, Madison, Wis.), comprising the 1.5 kb Hind III-Eco RI μenhancer fragment in the vector pAT153 (Amersham, Arlington Heights,Ill.), was digested with Hind III and Eco RI to isolate the 1.5 kbenhancer fragment. The adhesive ends of the enhancer fragment werefilled in by treatment with T4 DNA polymerase and deoxynucleotidetriphosphates. The blunt-ended fragment and pICμPR7, which had beenlinearized by digestion with Sma I, were joined by ligation. Theligation mixture was transformed into E. coli RR1. Plasmid DNA wasprepared from the transformants, and the plasmids were analyzed byrestriction digests. A plasmid comprising the μ enhancer and the μpromoter was designated pICμPRE8 (FIG. 7).

B. Construction of pSDL114

The DNA sequence encoding the extracellular domain of the PDGFβ-receptor was joined with the DNA sequence encoding the humanimmunoglobulin light chain constant region. The PDGF β-receptorextracellular domain was obtained from mpBTL22, which comprised the EcoRI-Hind III fragment from pBTL22 (Example 8.A.) cloned into Eco RI-HindIII cut M13mp18. Single stranded DNA was prepared from a mpBTL22 phageclone, and the DNA was subjected to in vitro mutagenesis using theoligonucleotide ZC1886 (Table 1) and the method described by Kunkel(Proc. Natl. Acad. Sci. USA 82: 488-492, 1985). A phage clone comprisingthe mutagenized PDGFβ-R with a donor splice site (5′ splice site) at the3′ end of the PDGFβ-R extracellular domain was designated pBTLR-HX (FIG.7).

The native PDGFβ-R signal sequence was obtained from pPR5. Plasmid pPR5,comprising 738 bp of 5′ coding sequence with an Eco RI site immediately5′ to the translation initiation codon, was constructed by in vitromutagenesis of the PDGFβ-R cDNA fragment from RP51 (Example 1).Replicative form DNA of RP51 was digested with Eco RI to isolate the1.09 kb PDGFβ-R fragment. The PDGFβ-R fragment was cloned into the EcoRI site of M13mp18. Single stranded template DNA was prepared from aphage clone containing the PDGFβ-R fragment in the proper orientation.The template DNA was subjected to in vitro mutagenesis usingoligonucleotide ZC1380 (Sequence ID Number 8; Table 1) and the methoddescribed by Zoller and Smith (Meth. Enzymol. 100: 468-500, 1983). Themutagenesis resulted in the placement of an Eco RI site immediately 5′to the translation initiation codon. Mutagenized phage clones wereanalyzed by dideoxy sequence analysis. A phage clone containing theZC1380 mutation was selected, and replicative form (Rf) DNA was preparedfrom the phage clone. The Rf DNA was digested with Eco RI and Acc I toisolate the 0.63 kb fragment. Plasmid pR-RXI (Example 1) was digestedwith Acc I and Eco RI to isolate the 3.7 kb fragment. The 0.63 kbfragment and the 3.7 kb fragment were joined by ligation resulting inplasmid pPR5 (FIG. 7).

As shown in FIG. 7, the PDGFβ-R signal peptide and part of theextracellular domain were obtained from plasmid pPR5 as a 1.4 kb EcoRI-Sph I fragment. Replicative form DNA from phage clone prBTLR-HX wasdigested with Sph I and Hind III to isolate the approximately 0.25 kbPDGFβ-R fragment. Plasmid pUC19 was linearized by digestion with Eco RIand Hind III. The 1.4 kb Eco RI-Sph I PDGFβ-R fragment, the 0.25 kb SphI-Hind III fragment from pBTLR-HX and the Eco RI-Hind III cut pUC19 werejoined in a three-part ligation. The resultant plasmid, pSDL110, wasdigested with Eco RI and Hind III to isolate the 1.65 kb PDGFβ-Rfragment.

Plasmid pICHuCκ3.9.11 was used as the source of the human immunoglobulinlight chain gene (FIG. 7). The human immunoglobulin light chain gene wasisolated from a human genomic library using an oligonucleotide probe (5′TGT GAC ACT CTC CTG GGA GTT A 3′; Sequence ID Number 32), which wasbased on a published human kappa C gene sequence (Hieter et al., Cell22: 197-207, : 1980). The human light chain (kappa) constant region wassubcloned as a 1.1 kb Sph I-Hind I genomic fragment of the human kappagene, which has been treated with DNA polymerase DNA I (Klenow Fragment)to fill in the Hind I adhesive end, into Sph I-Hind II cut pUC19. The1.1 kb human kappa constant region was subsequently isolated as a 1.1 kbSph I-Bam HI fragment that was subcloned into Sph I-Bgl II cut pIC19R(Marsh et al., ibid.). The resultant plasmid was designatedpICHuCλ3.9.11. Plasmid pICHuC_(κ)3.9.11 was digested with Hind III andEco RI to isolate the 1.1 kb kappa constant region gene. Plasmid pIC19Hwas linearized by digestion with Eco RI. The 1.65 kb PDGFβ-R fragment,the 1.1 kb human kappa constant region fragment and the linearizedpIC19H were joined in a three part ligation. The resultant plasmid,pSDL112, was digested with Bam HI and Cla I to isolate the 2.75 kbfragment. Plasmid pμPRE8 was linearized with Bgl II and Cla I. The 2.75kb fragment and the linearized pμPRE8 were joined by ligation. Theresultant plasmid was designated pSDL114 (FIG. 7).

Plasmid pSDL114 was linearized by digestion with Cla I and wascotransfected with Pvu I-digested p416 into SP2/0-Ag14 (ATCC CRL 1581)by electroporation using the method essentially described by Neumann etal. (EMBO J. 1: 841-845, 1982). (Plasmid p416 comprises the Adenovirus 5ori, SV40 enhancer, Adenovirus 2 major late promoter, Adenovirus 2tripartite leader, 5′ and 3′ splice sites, the DHFR^(r) cDNA, the SV40polyadenylation signal and pML-1 (Lusky and Botchan, Nature 293: 79-81,1981) vector sequences.) Transfectants were selected in growth mediumcontaining methotrexate.

Media from drug resistant clones were tested for the presence ofsecreted PDGF β-receptor analogs by enzyme-linked immunosorbant assay(ELISA). Ninety-six well assay plates were prepared by incubating 100 μlof 1 μg/ml polyclonal goat anti-human kappa chain (Cappel Laboratories,Melvern, Pa.) diluted in phosphate buffered saline (PBS; Sigma)overnight at 4° C. Excess antibody was removed by three washes with 0.5%Tween 20 in PBS. One hundred microliters of spent media was added toeach well, and the well were incubated for one hour at 4° C. Unboundproteins were removed by eight washes with 0.5% Tween 20 in PBS. Onehundred microliters of peroxidase-conjugated goat anti-human kappaantibody (diluted 1:1000 in a solution containing 5% chicken serum(GIBCO-BRL)+0.5% Tween 20 in PBS) was added to each well and the wellswere incubated for one hour at 4° C. One hundred microliters ofchromophore (100 μl ABTS (2,2′-Azinobis(3-ethylbenzthiazoline sulfonicacid) diammonium salt; Sigma)+1 μ/l 30% H₂O₂+12.5 ml citrate/phosphatebuffer (9.04 g/l citric acid, 10.16 g/l Na₂HPO₄)) was added to eachwell, and the wells were incubated to thirty minutes at roomtemperature. The samples were measured at 405 nm. The results of theassay showed that the PDGFβ-R analog secreted by the transfectantscontained an immunoglobulin light chain.

Spent media from drug resistant clones was also tested for the presenceof secreted PDGF β-receptor analogs by immunoprecipitation.Approximately one million drug resistant transfectants weremetabolically labeled by growth in DMEM medium lacking cysteine+2% calfserum for 18 hours at 37° C. in the presence of 50 μCI ³⁵S-cysteine.Media was harvested from the labeled cells and 250 μl of the spent mediawas assayed by immunoprecipitation with the anti-PDGF β-receptorantibody PR7212 to detect the presence of metabolically labeled PDGFβ-receptor analogs. PR7212, diluted in PBS, was added to the media to afinal concentration of 2.5 μg per 250 μl spent media. Five microlitersof rabbit anti-mouse Ig diluted in PBS was added to the PR7212/mediamixtures. The immunocomplexes were precipitated by the addition of 50 μl10% fixed Staph A (weight/volume in PBS). The immunocomplexes wereanalyzed on 8% SDS-polyacrylamide gels followed by autoradiographyovernight at −70° C. The results of the immunoprecipitation showed thatthe PDGF β-receptor analog secreted by the transfectants was bound bythe anti-PDGF β-receptor antibody. The combined results of the ELISA andimmunoprecipitation assays showed that the PDGF β-receptor analogsecreted by the transfectants contained both the PDGF β-receptorligand-binding domain and the human light chain constant region.

C. Cotransfection of pSDL114 with an Immunoglobulin Heavy Chain

Plasmid pSDL114 was cotransfected with pφ5V_(H)huCγ1M-neo, which encodesa neomycin resistance gene expression unit and a complete mouse/humanchimeric immunoglobulin heavy chain gene expression unit.

Plasmid pφ5V_(H)huCγ1M-neo was constructed as follows. The mouseimmunoglobulin heavy chain gene was isolated from a lambda genomic DNAlibrary constructed from the murine hybridoma cell line NR-ML-05(Serafini et al., Eur. J. Nucl. Med. 14: 232, 1988) using anoligonucleotide probe designed to span the V_(H)/D/J_(H) junction (5′GCA TAG TAG TTA CCA TAT CCT CTT GCA CAG 3′; Sequence ID Number 33). Thehuman immunoglobulin gamma-1 C gene was isolated from a human genomiclibrary using a cloned human gamma-4 constant region gene (Ellison etal., DNA 1: 11-18, 1981). The mouse immunoglobulin variable region wasisolated as a 5.3 kb Sst I-Hind III fragment from the original phageclone and the human gamma-1 C gene was obtained from the original phageclone as a 6.0 kb Hind III-Xho I fragment. The chimeric gamma-1 C genewas created by joining the V_(H) and C_(H) fragments via the common HindIII site and incorporating them with the E. coli neomycin resistancegene expression unit into pIC19H to yield pφ5V_(H)huCγ1M-neo.

Plasmid pSDL114 was linearized by digestion with Cla I and wasco-transfected into SP2/O-Ag14 cells with Asp 718 linearizedpφ5V_(H)huCγ1M-neo. The transfectants were selected in growth mediumcontaining methotrexate and neomycin. Media from drug-resistant cloneswere tested for their ability to bind PDGF in a competition bindingassay.

The competition binding assay measured the amount of ¹²⁵I-PDGF left tobind to human dermal fibroblast cells after preincubation with the spentmedia from pSDL114-pφ5V_(H)huCγ1M-neo transfected cells. The media wereserially diluted in binding medium (Table 4). The dilutions were mixedwith 0.5 ng of iodinated PDGF-BB or iodinated PDGF-AA, and the mixtureswere incubated for two hours at room temperature. Three hundredmicrograms of unlabeled PDGF-BB or unlabeled PDGF-AA was added to onetube from each series. The sample mixtures were added to 24 well platescontaining confluent human dermal fibroblast cells. The cells wereincubated with the mixture for four hours at 4° C., The supernatantswere aspirated from the wells, and the wells were rinsed three timeswith phosphate buffered saline that was held a 4° C. (PBS; Sigma, St.Louis, Mo.). Five hundred microliters of PBS+1% NP-40 was added to eachwell, and the plates were shaken on a platform shaker for five minutes.The cells were harvested and the amount of iodinated PDGF wasdetermined. The results of the competition binding assay showed that theprotein produced from pSDL114-pφ5V_(H)huCγ1M-neo transfected cells wasable to competitively bind PDGF-BB but did not bind PDGF-AA.

The PDGF β-receptor analog produced from a pSDL114-pφ5V_(H)huCγ1M-neotransfectant was assayed to determine if the receptor analog was able tobind PDGF-BB with high affinity. Eight and one half milliliters of spentmedia containing the PDGFβ-R analogs from a pSDL114-pφ5V_(H)huCγ1M-neotransfectant was added to 425 μl of Sepharose C1-4B-Protein A beads(Sigma, St. Louis, Mo.), and the mixture was incubated for 10 minutes at4° C. The beads were pelleted by centrifugation and washed with bindingmedium (Table 4). Following the wash the beads were resuspended in 8.5ml of binding media, and 0.25 ml aliquots were dispensed to 1.5 mltubes. Binding reactions were prepared by adding iodinated PDGF-BB_(Tyr)(Example 18.F.) diluted in DMEM+10% fetal calf serum to the identicalaliquots of receptor-bound beads to final PDGF-BB_(Tyr) concentrationsof between 4.12 pM and 264 pM. Nonspecific binding was determined byadding a 100 fold excess of unlabeled BB to an identical set of bindingreactions. Mixtures were incubated overnight at 4° C.

The beads were pelleted by centrifugation, and unbound PDGF-BB wasremoved with three washes in PBS. The beads were resuspended in 100 μlof PBS and were counted. Results of the assay showed that the PDGFβ-Ranalog was able to bind PDGF-BB with high affinity.

D. Construction of pSDL113

As shown in FIG. 8, the DNA sequence encoding the extracellular domainof the PDGF β-receptor was joined with the DNA sequence encoding a humanimmunoglobulin heavy chain constant region joined to a hinge sequence.Plasmid pSDL110 was digested with Eco RI and Hind III to isolate the1.65 kb PDGFβ-R fragment. Plasmid pICHuγ-1M was used as the source ofthe heavy chain constant region and hinge region. Plasmid pICHuγ-1Mcomprises the approximately 6 kb Hind III-Xho I fragment of a humangamma-1 C gene subcloned into pUC19 that had been linearized bydigestion with Hind III and SaI I. Plasmid pICHuγ-1M was digested withHind III and Eco RI to isolate the 6 kb fragment encoding the humanheavy chain constant region. Plasmid pIC19H was linearized by digestionwith Eco RI. The 1.65 kb PDGFβ-R fragment, the 6 kb heavy chain constantregion fragment and the linearized pIC19H were joined in a three partligation. The resultant plasmid, pSDL111, was digested with Bam HI toisolate the 7.7 kb fragment. Plasmid pμPRE8 was linearized with Bgl IIand was treated with calf intestinal phosphatase to preventself-ligation. The 7.7 kb fragment and the linearized pμPRE8 were joinedby ligation. A plasmid containing the insert in the proper orientationwas designated pSDL113 (FIG. 8).

Plasmid pSDL113 is linearized by digestion with Cla I and iscotransfected with Pvu I-digested p416 into SP2/0-Ag14 byelectroporation. Transfectants are selected in growth medium containingmethotrexate.

Media from drug resistant clones are tested for the presence of secretedPDGFβ-R analogs by immunoprecipitation using the method described inExample 12.B.

E. Cotransfection of pSDL113 with an Immunoglobulin Light Chain Gene

Plasmid pSDL113 is linearized by digestion with Cla I and wascotransfected with pICφ5V_(κ)HuC_(κ)-Neo, which encodes a neomycinresistance gene and a mouse/human chimeric immunoglobulin light chaingene. The mouse immunoglobulin light chain gene was isolated from alambda genomic DNA library constructed from the murine hybridoma cellline NR-ML-05 (Woodhouse et al., ibid.) using an oligonucleotide probedesigned to span the V_(κ)/J_(κ) junction (5′ ACC GAA CGT GAG AGG AGTGCT ATA A 3′; Sequence ID Number 34). The human immunoglobulin lightchain constant region gene was isolated as described in Example 12.B.The mouse NR-ML-05 immunoglobulin light chain variable region gene wassubcloned from the original mouse genomic phage clone into pIC19R as a 3kb Xba I-Hinc II fragment. The human kappa C gene was subcloned from theoriginal human genomic phage clone into pUC19 as a 2.0 kb Hind III-EcoRI fragment. The chimeric kappa gene was created by joining the NR-ML-05light chain variable region gene and human light chain constant regiongene via the common Sph I site and incorporating them with the E. colineomycin resistance gene into pIC19H to yield pICφ5V_(κ)HuC_(κ)-Neo(FIG. 9).

The linearized pSDL113 and pICφ5V_(κ)HuC_(κ)-Neo are transfected intoSP2/0-Ag14 cells, by electroporation. The transfectants are selected ingrowth medium containing methotrexate and neomycin.

F. Cotransfection of pSDL113 and pSDL114

A clone of SP2/0-Ag14 stably transfected with pSDL114 and p416 wasco-transfected with Cla I-digested pSDL113 and Bam HI-digested pICneo byelectroporation. (Plasmid pICneo comprises the SV40 promoter operativelylinked to the E. coli neomycin resistance gene and pIC19H vectorsequences.) Transfected cells were selected in growth medium containingmethotrexate and G418. Media from drug-resistant clones were tested fortheir ability to bind PDGF-BB or PDGF-AA in a competition binding assayas described in Example 12.C. The results of the assay showed that thetransfectants secreted a PDGF β-receptor analog which was capable ofcompetitively binding PDGF-BB but did not detectably bind to PDGF-AA.

G. Cotransfection of pSDL114 with Fab

A clone of SP2/0-AG14 stably transfected with pSDL114 and p416 wastransfected with the Fab region of the human gamma-4 gene (γ4) inplasmid pφ5V_(H)Fab-neo.

Plasmid pφ5V_(H)Fab-neo was constructed by first digesting plasmidp24BRH (Ellison et al., DNA 1: 11, 1988) was digested with Xma I and EcoRI to isolate the 0.2 kb fragment comprising the immunoglobulin 3′untranslated region. Synthetic oligonucleotides ZC871 (Sequence IDNumber 3; Table 1) and ZC872 (Sequence ID Number 4; Table 1) werekinased and annealed using essentially the methods described by Maniatiset al. (ibid.). The annealed oligonucleotides ZC871/ZC872 formed an SstI-Xma I adapter. The ZC871/ZC872 adapter, the 0.2 kb p24BRH fragment andSst I-Eco RI linearized pUC19 were joined in a three-part ligation toform plasmid Pγ₄3′. Plasmid Pγ₄3′ was linearized by digestion with BamHI and Hind III. Plasmid p24BRH was cut with Hind III and Bgl II toisolate the 0.85 kb fragment comprising the C_(H)1 region. The pγ₄3′fragment and the Hind III-Bgl II p24BRH fragment were joined by ligationto form plasmid pγ₄Fab. Plasmid pγ₄Fab was digested with Hind III andEco RI to isolate the 1.2 kb fragment comprising γ₄Fab. Plasmid pICneo,comprising the SV40 promoter operatively linked to the E. coli neomycinresistance gene and pIC19H vector sequences, was linearized by digestionwith Sst I and Eco RI. Plasmid pφ5V_(H), comprising the mouseimmunoglobulin heavy chain gene variable region and pUC18 vectorsequences, was digested with Sst I and Hind III to isolate the 5.3 kbV_(H) fragment. The linearized pICneo was joined with the 5.3 kb SstI-Hind III fragment and the 1.2 kb Hind III-Eco RI fragment in athree-part ligation. The resultant plasmid was designatedpφ5V_(H)Fab-neo (FIG. 10).

A pSDL114/p416-transfected SP2/0-AG14 clone was transfected with ScaI-linearized pφ5V_(H)Fab-neo. Transfected cells were selected in growthmedium containing methotrexate and G418. Media from drug-resistantclones were tested for their ability to bind PDGF in a competitionbindings assay as described in Example 12.C. The results of the assayshowed that the PDGF β-receptor analog secreted from the transfectantswas capable of competitively binding PDGF-BB.

H. Cotransfection of pSDL114 with Fab′

A stably transfected SP2/0-AG14 isolate containing pSDL114 and p416 wastransfected with plasmid pWKI, which contained the Fab′ portion of animmunoglobulin heavy chain gene. Plasmid pWKI was constructed asfollows.

The immunoglobulin gamma-1 Fab′ sequence, comprising the C_(H)1 andhinge regions sequences, was derived from the gamma-1 gene clonedescribed in Example 12.C. The gamma-l gene clone was digested with HindIII and Eco RI to isolate the 3.0 kb fragment, which was subcloned intoHind III-Eco RI linearized M13mp19. Single-stranded template DNA fromthe resultant phage was subjected to site-directed mutagenesis usingoligonucleotide ZC1447 (Sequence ID Number 9; Table 1) and essentiallythe method of Zoller and Smith (ibid.). A phage clone was identifiedhaving a ZC1447 induced deletion resulting in the fusion of the hingeregion to a DNA sequence encoding the amino acidsAla-Leu-His-Asn-His-Tyr-Thr-Glu-Lys-Ser-Leu-Ser-Leu-Ser-Pro-Gly-Lys(Sequence ID Number 31) followed in-frame by a stop codon. Replicativeform DNA from a positive phage clone was digested with Hind III and EcoRI to isolate the 1.9 kb fragment comprising the C_(H)1 and hingeregions. Plasmid pφ5V_(H) was digested with Sst I and Hind III toisolate the 5.3 kb fragment comprising the mouse immunoglobulin heavychain gene variable region. Plasmid pICneo was linearized by digestionwith Sst I and Eco RI. The linearized pICneo was joined with the 5.3 kbHind III-Sst I fragment and the 1.9 kb Hind III-Eco RI fragment in athree-part ligation. The resultant plasmid was designated pWKI (FIG.10).

An SP2/0-AG14 clone stably transfected with pSDL114 and p416 wastransfected with Asp 718-linearized pWKI. Transfected cells wereselected by growth in medium containing methotrexate and G418. Mediasamples from transfected cells were assayed using the competition assaydescribed in Example 12.C. Results from the assays showed that thetransfected cells produced a PDGF β-receptor analog capable ofcompetitively binding PDGF-BB.

Example 13 Purification and Characterization of PDGF β-Receptor Analogsfrom Mammalian cells Co-transfected With pSDL113 and pSDL114

A. Purification of PDGF β-Receptor Analogs

The PDGF β-receptor analog was purified from conditioned culture mediafrom a clone of transfected cells grown in a hollow fiber system. Themedia was passed over a protein-A sepharose column, and the column waswashed sequentially with phosphate buffered saline, pH 7.2 (PBS; Sigma,St. Louis, Mo.) and 0.1 M citrate, pH 5.0. The PDGF β-receptor analogwas eluted from the protein-A column with 0.1 M citrate pH 2.5 andimmediately neutralized by the addition of Tris-base, pH 7.4. The eluatefractions containing PDGF β-receptor analog, as determined by silverstain, were pooled and chromatographed over an S-200 column (PharmaciaLKB Technologies, Inc., Piscataway, N.J.) equilibrated with PBS. Thepeak fractions from the S-200 column were pooled and concentrated on acentriprep-10 concentrator (Amicon). Glycerol (10% final volume) wasadded to the preparation and the sample frozen at −80° C. PDGFβ-receptor analogs purified from pSDL114+pSDL113 co-transfected cellswere termed “tetrameric PDGF α-receptors”.

B. Measurement of the Relative Binding Affinity of Tetrameric PDGFβ-Receptor Analog by Soluble Receptor Assay

Purified tetrameric PDGF β-receptor analog was compared to detergentsolubilized extracts of human dermal fibroblasts for ¹²⁵I-labeledPDGF-BB binding activity in a soluble receptor assay essentially asdescribed by Hart et al. (J. Biol. Chem. 262: 10780-107:35, 1987). Humandermal fibroblast cells were extracted at 20×10⁶ cell equivalents per mlin TNEN extraction buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mMEDTA, 0.5% Nonidet P-40, 1 mM PMSF, 10% glycerol). Two hundred and fiftythousand PDGF β-receptor-subunits per cell was used to calculate thetetrameric PDGF β-receptor analog number per volume of extract. Thisvalue has been previously published by Seifert et al. (J. Biol Chem.264: 8771-8778, 1989). The PDGF β-receptor analog number was determinedfrom the protein concentration of the PDGF β-receptor analog assuming anaverage molecular weight of 140 kDa for each immunoglobulin-PDGFβ-receptor monomer, and four monomers per tetramer. Thus, eachtetrameric molecule contains four receptor molecules.

Increasing amounts of either detergent solubilized extracts of humandermal fibroblast cells or purified PDGF β-receptor analog wereincubated with 1 ng of ¹²⁵I-labeled PDGF-BB for one hour at 37° C. Thesample was then diluted with 1 ml binding media and was added tomonolayers of human dermal fibroblast cells grown in 24-well culturedishes. The samples were incubated for two hours at 4° C. The wells werewashed to remove unbound, ¹²⁵I-labeled PDGF-BB. On half of a milliliterof extraction buffer (PBS+1% Nonidet P-40) was added to each wellfollowed by a 5 minute incubation. The extraction mixtures wereharvested and counted in a gamma counter.

The results showed that the PDGF β-receptor analog had the same relativebinding affinity as solubilized PDGF β-receptor-subunit from mammaliancells in a solution phase binding assay.

C. Determination of the Binding Affinity of the PDGF β-Receptor Analogin a Solid Phase Format

The apparent dissociation constant K_(D)(app) of the PDGF β-receptoranalog was determined essentially as described by Bowen-Pope and Ross(Methods in Enzymology 109: 69-100, 1985), using the concentration of¹²⁵I-labeled PDGF-BB giving half-maximal specific ¹²⁵I-labeled PDGF-BBbinding. Saturation binding assays to determine the concentration of¹²⁵I-labeled PDGF-BB that gave half-maximal binding to immobilized PDGFβ-receptor analog were conducted as follows.

Affinity purified goat anti-human IgG, H- and L-chain (Commerciallyavailable from Cappel Labs) was diluted into 0.1 M Na₂HCO₃, pH 9.6 to aconcentration of 2 μg/ml. One hundred microliters of the antibodysolution was coated onto each well of 96-well microtiter plates for 18hours at 4° C. The wells were washed once with ELISA C buffer (PBS+0.05%Tween-20) followed by an incubation with 175 μl/well of ELISA B buffer(PBS+1% BSA+0.05% Tween-20) to block the wells. The wells were washedonce with ELISA B buffer. One hundred microliters of 12.1 ng/ml or 24.3ng/ml of tetrameric PDGF β-receptor analog protein diluted in ELISA Bwas added to each well and the plates were incubated for 2 hours at 37°C. Unbound protein was removed from the wells by two washes with ELISAC. ¹²⁵I-labeled PDGF-BB_(Tyr) (Example 18.F.) was serially diluted intobinding media (25 mM HEPES, pH 7.2, 0.25% rabbit serum albumin dilutedin HAMs F-12 medium (GIBCO-BRL)), and 100 μl of the dilutions were addedto the wells. The plates were incubated for two hours at roomtemperature. The unbound ¹²⁵I-labeled PDGF-BB was removed, and the wellswere washed three times with binding media. Following the last wash, 100μl of 0.1 M citrate, pH 2.5 was added to each well. After five minutes,the citrate buffer was removed, transferred to a tube and counted in agamma counter. The counts reflect counts of ¹²⁵I-labeled PDGF-BB_(Tyr)bound by the receptor analog. Nonspecific binding for each concentrationof ¹²⁵I-labeled PDGF-BB_(Tyr) was determined by a parallel assay whereinseparate wells coated only with goat anti-human IgG were incubated withthe ¹²⁵I-labeled PDGF-BB concentrations. Nonspecific binding wasdetermined to be 2.8% of the total input counts per well and averaged 6%of the total counts bound.

Saturation binding assay on 12.1 and 24.3 ng/ml of tetrameric PDGFβ-receptor analog gave half-maximal binding at 0.8 and 0.82 ng/ml¹²⁵I-labeled PDGF-BB_(Tyr), respectively. By Scatchard analysis(Scatchard, Ann. NY Acad. Sci 51: 660-667, 1949) these values were shownto correspond to a K_(D)(app) of 2.7×10⁻¹¹ which agree with thepublished values for PDGF receptors on mammalian cells.

Example 14 Solid Phase Ligand Binding Assay Using the PDGF β-ReceptorAnalog

A. Solid Phase Radioreceptor Competition Binding Assay

In a solid phase radioreceptor competition binding assay (RRA), thewells of 96-well microtiter plates were coated with 100 μl of 2 μg/mlaffinity purified goat anti-human IgG (Cappel Labs) diluted in 0.1 MNa₂HCO₃, pH 9.6. After an eighteen hour incubation at 4° C., the wellswere washed once with ELISA C. The wells were blocked by incubation for2 hours at 37° C. with 175 μl/well ELISA B. The wells were washed oncewith ELISA B then incubated for 2 hours at 37° C. with 50 ng/mltetrameric PDGF β-receptor analog diluted in ELISA B. The unboundreceptor was removed, and the test wells were incubated with increasingconcentrations of serially diluted, unlabeled PDGF-BB (diluted inbinding media. Following a two hour incubation at room temperature, thewells were washed three times with binding media. One hundredmicroliters of 5 ng/ml ¹²⁵I-labeled PDGF-BB_(Tyr) (Example 18.F.) wasadded to each well, and the plates were incubated for an additional twohours at room temperature. The wells were washed three times withbinding media followed by a 5 minute incubation with 100 μl/well of 0.1M citrate, pH 2.5. The samples were harvested and counted in a gammacounter.

Radioreceptor assay (RRA) competition binding curves were generated forPDGF β-receptor analog protein plated at 48.6 ng/ml. The sensitivity ofthe assays is 1 ng/ml of PDGF-BB, with 8 ng/ml giving 50% inhibition in¹²⁵I-PDGF-BB binding, and a working range between 1 and 32 ng/ml ofPDGF-BB. The values were similar to those obtained using monolayers ofSK-5 cells in an RRA.

B. Use of Tetrameric PDGF β-Receptor Analogs As Antagonists forPDGF-Stimulated Mitogenesis

A tetrameric PDGF β-receptor analog, purified as described in Example13, was analyzed for the ability to neutralize PDGF-stimulatedmitogenesis in mouse 3T3 cells. Increasing amounts of the purifiedtetrameric PDGF β-receptor analog were mixed with 5 ng of PDGF. Themixtures were then added to cultures of mouse 3T3 cells. The ability ofthe PDGF to stimulate a mitogenic response, as measured by theincorporation of ³H-thymidine, was determined essentially as described(Raines and Ross, Methods in Enzymology 109: 749-773, 1985, which isincorporated by reference herein). The tetrameric PDGF β-receptor analogdemonstrated a dose response inhibition of PDGF-BB-stimulated³H-thymidine incorporation, while having essentially no effect onPDGF-AA- and PDGF-AB-stimulated ³H-thymidine incorporation.

C. Binding of Tetrameric PDGF β-receptor Analog to Immobilized PDGF

A tetrameric PDGF β-receptor analog, purified as described in Example13, was analyzed for its ability to bind to immobilized PDGF. PDGF-BB(100 ng/ml) was coated onto wells a 96-well microtiter plate, and theplates were incubated 18 hours at 4° C. followed by one wash with ELISAC buffer. The wells were incubated for 2 hours 37° C. with ELISA Bbuffer to block the wells. Increasing concentrations of ¹²⁵I-labeledtetrameric PDGF β-receptor analog, diluted in binding media, was addedto the wells for two hours at room temperature. The wells were washedfour times with ELISA C buffer to remove unbound receptor analog. Onehundred microliters of 1 M H₂SO₄ was added to each well and the plateswere incubated for five minutes at room temperature. The solution wasthen harvested and transferred to tubes to be counted in a gammacounter. Nonspecific binding was determined to be less than 10% of thetotal counts bound.

A receptor competition binding assay was developed using this assayformat. The assay was carried out as described above, and simultaneousto the addition of the ¹²⁵I-labeled tetrameric PDGF β-receptor analog,increasing amounts of PDGF-AA, AB or BB were added to the PDGF-BB coatedwells. Under these conditions, only PDGF-BB was found to significantlyblock the binding of the labeled PDGF β-receptor analog to theimmobilized PDGF-BB.

Example 15 Construction and Expression of PDGFα-R Analogs in CulturedMouse Myeloma Cells

A. Construction of an Optimized PDGFα-R cDNA

The PDGF α-receptor coding region was optimized for expression inmammalian cells as follows. The 5′ end of the cDNA was modified toinclude an optimized Kozak consensus translation initiation sequence(Kozak, Nuc. Acids Res. 12: 857-872, 1984) and Eco RI and Bam HI sitesjust 5′ of the initiation methionine codon. Oligonucleotides ZC2181,ZC2182, ZC2183 and ZC2184 (Sequence ID Numbers 23, 24, 25 and 26,respectively; Table 1) were designed to form, when annealed, an adapterhaving an Eco RI adhesive end, a Bam HI restriction site, a sequenceencoding a Kozak consensus sequence 5′ to the initation methioninecodon, a mammalian codon optimized sequence encoding amino acids 1-42 ofFIGS. 11A-11D, and an Eco RI adhesive end that destroys the Eco RI sitewithin the PDGFα-R coding sequence. The adapter also introduced adiagnostic Cla I site 3′ to the initiation methionine codon.Oligonucleotides ZC2181, ZC2182, ZC2183 and ZC2184 were kinased,annealed and ligated. Plasmid pα17B was linearized by partial digestionwith Eco RI. The linearized pα17B was ligated with theZC2181/ZC2182/ZC2183/ZC2184 oligonucleotide adapter, and the ligationmixture was transformed into E. coli. Plasmid DNA prepared from thetransformants was analyzed by restriction analysis and a positive clonehaving the oligonucleotide adapter in the correct orientation wasdigested with Eco RI and Pst I to isolate the 1.6 kb fragment. Thisfragment was subcloned into Eco RI+Pst I-linearized M13mp19. Theresultant phage clone was designated 792-8. Single-stranded 792-8 DNAwas sequenced to confirm the orientation of the adapter.

A fragment encoding the ligand-binding domain of the PDGF α-receptor(PDGFα-R) was then generated as follows. Restriction sites and a splicedonor sequence were introduced at the 3′ end of the PDGFα-Rextracellular domain by PCR amplification of the 792-8 DNA andoligonucleotides ZC2311 and ZC2392 (Sequence ID Numbers 27 and 30, Table1). Oligonucleotide ZC2311 is a sense primer encoding nucleotides 1470to 1489 of FIGS. 11A-11D. Oligonucleotide ZC2392 is an antisense primerthat encodes nucleotides 1759 to 1776 of FIGS. 11A-11D followed by asplice donor and Xba I and Hind III restriction sites. The 792-8 DNA wasamplified using manufacturer recommended (Perkin Elmer Cetus, Norwalk,CT) conditions and the GeneAmp™ DNA amplification reagent kit (PerkinElmer Cetus), and blunt-ended 329 bp fragment was isolated. Theblunt-end fragment was digested with Nco I and Hind III and ligated withSma I-digested pUC18. A plasmid having an insert with the Nco I sitedistal to the Hind III site present in the pUC18 polylinker wasdesignated pUC18 Sma-PCR Nco HIII #13. The Hind III site present in theinsert was not regenerated upon ligation with the linearized pUC18.Plasmid pUC18 Sma-PCR Nco HIII #13 was digested with Nco I and Hind IIIto isolate the 555 bp PDGFα-R containing fragment encoding PDGFαR.Oligonucleotides ZC2351 and ZC2352 (Table 1; Sequence ID Numbers 28 and29) were kinased and annealed to form an Sst I-Nco I adapter encoding aninternal Eco RI site and a Kozak consensus translation initiation site.The 355 bp Nco I-Hind III fragment, the ZC2351/ZC2352 adapter and a 1273bp Nco I fragment comprising the extracellular domain of PDGF α-Rderived from 792-8 were ligated with Hind III+SstI-digested pUC18 andtransformed into E. coli. Plasmid DNA was isolated from thetransformants and analyzed by restriction analysis. None of the isolatescontained the 1273 bp Nco I fragment. A plasmid containing the NcoI-Hind III fragment and the ZC2351/ZC2352 adapter was designated pUC18Hin Sst Δ Nco #46. Plasmid pUC18 Hin Sst Δ Nco #46 was linearized bydigestion and joined by ligation with the 1273 bp Nco I fragmentcomprising the extracellular domain of the PDGFα-R from clone α18 R-19.The ligations were transformed into E. coli, and plasmid DNA wasisolated from the transformants. Analysis of the plasmid DNA showed thatonly clones with the Nco I fragment in the wrong orientation wereisolated. A clone having the Nco I fragment in the wrong orientation wasdigested with Nco I, religated and transformed into E. coli. Plasmid DNAwas isolated from the transformants and was analyzed by restrictionanalysis. A plasmid having the Nco I insert in the correct orientationwas digested to completion with Hind III and partially digested with SstI to isolate the 1.6 kb fragment comprising the extracellular domain ofthe PDGFα-R preceded by a consensus initiation sequence (Kozak, ibid.)and followed by a splice donor site.

B. Construction of pPAB7

The DNA sequence encoding the extracellular domain of the PDGFα-R wasjoined to the immunoglobulin μ enhancer-promoter and to a DNA sequenceencoding an immunoglobulin light chain constant region. Theimmunoglobulin μ enhancer-promoter was obtained from plasmid pJH1 whichwas derived from plasmid PICμPRE8 (Example 12.A.) by digestion with EcoRI and Sst I to isolate the 2.2 kb fragment comprising theimmunoglobulin enhancer and heavy chain variable region promoter. The2.2 kb Sst I-Eco RI fragment was ligated with Sst I+Eco RI-linearizedpUC19. The resulting plasmid, designated pJH1, contained theimmunoglobulin enhancer and heavy chain variable region promoterimmediately 5′ to the pUC19 linker sequences. Plasmid pJH1 waslinearized by digestion with Sst I and Hind III and joined with the 1.6kb partial Sst I-Hind III fragment containing the PDGFα-R extracellulardomain sequences. The resulting plasmid having the immunoglobulin μenhancer-promoter joined to the PDGFα-R extracellular domain wasdesignated pPAB6. Plasmid pSDL112 was digested with Hind III to isolatethe 1.2 kb fragment encoding the immunoglobulin light chain constantregion (Cκ). The 1.2 kb Hind III fragment was ligated with HindIII-linearized pPAB6. A plasmid having the C_(κ) sequence in the correctorientation was designated pPAB7.

C. Construction of pPAB9

The partial Sst I-Hind III fragment encoding the extracellular domain ofthe PDGFα-R was joined to the immunoglobulin heavy chain constantregion. For convenience, the internal Xba I site in plasmid pJH1 wasremoved by digestion with Xba I, blunt-ending with T4 DNA polymerase,and religation. A plasmid which did not contain the internal Xba I site,but retained the Xba I site in the polylinker was designated 11.28.3.6.Plasmid 11.28.3.6 was linearized by digestion with Sst I and Xba I.Plasmid pPAB6 was digested to completion with Hind III and partiallydigested with Sst I to isolate the 1.6 kb Sst I-Hind III fragmentcontaining the PDGFα-R extracellular domain. Plasmid pφ5V_(H)huCγ1M-neo(Example 12.C.) was digested with Hind III and Xba I to isolate the 6.0kb fragment encoding the immunoglobulin heavy chain constant region(huCγ1M). The Sst I-Hind III-linearized 11.28.3.6, the 1.6 kb SstI-Hindi III PDGFα-R fragment and the 6.0 kb Hind III-Xba I huCγ1Mfragment were ligated to form plasmid pPAB9.

D. Expression of pPAB9 in Mammalian Cells

Bgl II-linearized pPAB7 and Pvu I-linearized pPAB9 were cotransfectedwith Pvu I-linearized p416 into SP2/0-Ag14 cells by electroporation.Transfected cells were initially selected in growth medium containing 50nM methotrexate and were subsequently amplified in a growth mediumcontaining 100 μM methotrexate. Media from drug resistant clones weretested for the presence of secreted PDGF α-receptor analogs byenzyme-linked immunosorbant assay (ELISA). Ninety-six well assay plateswere prepared by incubating 100 μl of 1 μg/ml monoclonal antibody292.1.8 which is specific for the PDGF α-receptor diluted in phosphatebuffered saline (PBS; Sigma] overnight at 4° C. Excess antibody wasremoved by three washes with 0.5% Tween 20 in PBS. One hundredmicroliters of spent media was added to each well, and the plates wereincubated for one hour at 4° C. Unbound proteins; were removed by eightwashes with 0.5% Tween 20 in PBS. One hundred microliters ofperoxidase-conjugated goat anti-human IgG heavy chain antibody (diluted1:1000 in a solution containing 5% chicken serum (GIBCO-BRL)+0.5% Tween20 in PBS) was added to each well, and the plates were incubated for onehour at 4° C. One hundred microliters of chromophore (100 μl ABTS[2,2′-Azinobis(3-ethylbenz-thiazoline sulfonic acid] diammonium salt;Sigma]+1 μl 30% H₂O₂ +12.5 ml citrate/phosphate buffer [9.04 g/l citricacid, 10.16 g/l Na₂HPO₄]) was added to each well, and the wells wereincubated for 30 minutes at room temperature. The samples were measuredat 405 nm. The results of the assay showed that the PDGF α-receptoranalogs secreted by the transfectants contained an immunoglobulin heavychain.

Analysis of spent media from transfected cells by Northern analysis,Western analysis and by radioimmunoprecipitation showed that thetransfectants did not express a PDGF α-receptor analog from the pPAB7construction. Transfectants were subsequently treated as containing onlypPAB9.

Drug resistant clones were also tested for the presence of secreted PDGFα-receptor analogs by immunoprecipitation. For each clone, approximatelyone million drug resistant transfectants were grown in DMEM lackingcysteine+2% calf serum for 18 hours at 37° C. in the presence of 50 μCi³⁵S-cysteine. The spent media was harvested from the labeled cells and250 μl of medium from each clone was assayed for binding to theanti-PDGF α-receptor antibody 292.18. Monoclonal antibody 292.18 dilutedin PBS was added to each sample to a final concentration of 2.5 μg per250 μl spent media. Five microliters of rabbit anti-mouse Ig diluted inPBS was added to each sample, and the immunocomplexes were precipitatedby the addition of 50 μl 10% fixed Staph A (weight/volume in PBS). Theimmunocomplexes were analyzed on 8% SDS-polyacrylamide gels followed byautoradiography overnight at −70° C. The results of theimmunoprecipitation showed that the PDGF α-receptor analog secreted bythe transfectants was bound by the anti-PDGF α-receptor antibody. Thecombined results of the ELISA and immunoprecipitation assays showed thatthe PDGF α-receptor analog secreted by the transfectants contained boththe PDGF α-receptor ligand-binding domain and the human heavy chain.

Spent medium from drug-resistant clones were tested for their ability tobind PDGF in a competition binding assay essentially as described inExample 12.C. The results of the assay showed that the transfectantssecreted a PDGF α-receptor analog capable of binding PDGF-AA. A clonecontaining the pPAB9 was designated 3.17.1.57.

E. Co-expression of pPAB7 and pPAB9 in Mammalian Cells

Bgl II-linearized pPAB7 and Bam HI-linearized pICneo were cotransfectedinto clone 3.17.1.57, and transfected cells were selected in thepresence of neomycin. Media from drug resistant cells were assayed forthe presence of immunoglobulin heavy chain, immunoglobulin light chainand the PDGF α-receptor ligand-binding domain by ELISA essentially asdescribed above. Briefly, ninety-six well assay plates were prepared byincubating 100 μl of 1 μg/ml goat anti-human IgG Fc antibody (Sigma) or100 μl of 1 μg/ml 292.18 overnight at 4° C. Excess antibody was removedby three washes with 0.5% Tween 20 in PBS. One hundred microliters ofspent media was added to each well of each plate, and the plates wereincubated for one hour at 4° C. Unbound proteins were removed by eightwashes with 0.5% Tween 20 in PBS. One hundred microliters ofperoxidase-conjugated goat anti-human IgG antibody (diluted 1:1000 in asolution containing 5% chicken serum (GIBCO-BRL)+0.5% Tween 20 in PBS)was added to each well of the plate coated with the anti-Fc antibody,and 100 μl of peroxidase-conjugated goat anti human kappa antibody(diluted 1:1000 in a solution containing 5% chicken serum(GIBCO-BRL)+0.5% Tween 20 in PBS) was added to each well of the platecoated with 292.18. The plates were incubated for one hour at 4° C. Onehundred microliters of chromophore (100 μl ABTS[2,2′-Azinobis(3-ethylbenz-thiazoline sulfonic acid) diammonium salt;Sigma]+1 μl 30% H₂O₂+12.5 ml citrate/phosphate buffer [9.04 g/l citricacid, 10.16 g/l Na₂HPO₄]) was added to each well of each plate, and theplates were incubated to 30 minutes at room temperature. The sampleswere measured at 405 nm, the wavelength giving maximal absorbance of thechromogenic substrate, to identify clones having absorbances higher thanbackground indicating the presence of immunoglobulin heavy chain. Clonesthat gave positive results in both ELISA assays (showing that the clonesproduced proteins containing heavy chain regions, light chain constantregions and the PDGF α-receptor ligand-binding region) were selected forfurther characterization.

Drug resistant clones, that were positive for both ELISA assays weresubsequently tested for the presence of secreted PDGF α-receptor analogsby immunoprecipitation. For each positive clone, approximately onemillion drug resistant transfectants were grown in DMEM lackingcysteine+2% calf serum for 18 hours at 37° C. in the presence of 50 μCI³⁵S-cysteine. The spent media was harvested from the labeled cells and250 μl of medium from each clone was assayed for binding to monoclonalantibody 292.18. Monoclonal antibody 292.18 diluted in PBS was added toeach sample to a final concentration of 2.5 μg. Five microliters ofrabbit anti-mouse Ig diluted in PBS was added to each sample and theimmunocomplexes were precipitated by the addition of 50 μl 10% fixedStaph A (weight/volume in PBS). The immunocomplexes were analyzed on 8%SDS-polyacrylamide gels followed by autoradiography overnight at −70° C.The results of the immunoprecipitation showed that the PDGF α-receptoranalog secreted by the transfectants was bound by the anti-PDGFα-receptor antibody. The combined results of the ELISA andimmunoprecipitation assays showed that the PDGF α-receptor analogsecreted by the transfectants contained the PDGF α-receptorligand-binding domain, the human heavy chain and the human light chainconstant region. A clone that secreted a PDGF α-receptor analog that waspositive for both the above-described ELISA assays and theimmunoprecipitation assay was designated 5.6.2.1.

Example 16 Purification and Characterization of PDGF α-Receptor Analogs

A. Purification of PDGF α-Receptor Analogs From Clone 3.17.1.57

The PDGF α-receptor analog was purified from the conditioned culturemedia of clone 3.17.1.57 by cycling cell-conditioned medium over animmunoaffinity column composed of monoclonal antibody 2932.18 bound to aCNBr-activated Sepharose 4B resin, which is specific for the PDGFα-receptor. The column was washed with PBA, then eluted with 0.1 Mcitrate, pH 3.0. The peak column fractions containing the α-receptorwere pooled, neutralized to pH 7.2 by the addition of 2 M Tris, pH 7.4,then passed over a protein-A Sepharose column. This column was washedsequentially with PBS, then with 0.1 M citrate, pH 5.0. The PDGFα-receptor analog was then eluted with 0.1 M citrate, pH 3.0. The peakeluate fractions were pooled, and glycerol was added to a finalconcentration of 10%. The sample was concentrated on a centriprep 10concentrator (Amicon). The PDGF α-receptor analog purified from clone3.17.157 was termed a “dimeric PDGF α-receptor analog”.

B. Purification of PDGF α-Receptor Analogs From Clone 5.6.2.1

The PDGF α-receptor analog was purified from the conditioned culturemedia of clone 5.6.2.1 by cycling cell-conditioned medium over theimmunoaffinity column described above. The column was washed with PBSthen eluted with 0.1 M citrate, pH 3.0. The peak column fractionscontaining the α-receptor were pooled, neutralized to pH 7.2 by theaddition of 2 M Tris (what pH 7.4), then passed over a protein-Asepharose column. This column was washed sequentially with PBS then with0.1 M citrate, pH 5.0. The PDGF α-receptor analog was then eluted with0.1 M citrate, pH 3.0. The peak eluate fractions were pooled andglycerol was added to a final concentration of 10%. The sample wasconcentrated on a centriprep 10 concentrator. The PDGF α-receptoranalogs purified from clone 5.6.2.1 was termed a “tetrameric PDGFα-receptor analog”.

Example 17

A. Use of the PDGF α-receptor Analogs in Ligand Binding Studies

Purified tetrameric PDGF α-receptor analog and purified dimeric PDGFα-receptor analog were compared to monolayers of a control cell line ofcanine kidney epithelial cells, which do not naturally express the PDGFα-receptor, transfected with the human PDGF α-receptor cDNA for ligandbinding activity. The dissociation constant (Kd) of the receptorpreparations was determined by saturation binding and subsequentScatchard analysis.

Ligand binding of the purified PDGF α-receptor analogs was determinedusing a solid phase binding assay. Affinity-purified goat anti-human IgGwas diluted to a concentration of 2 μg/ml in 0.1 M Na₂HCO₃, pH 9.6 and100 μl/well of the solution was used to coat 96-well microtiter platesfor 18 hours at 4° C. Excess antibody was removed from the wells withone wash with ELISA C buffer (PBS, 0.05% Tween-20). The plates wereincubated with 175 μl/well of ELISA B buffer (PBS, 1% BSA, 0.05%Tween-20) to block the wells, followed by two washes with ELISA Cbuffer. One hundred microliters of 50 ng/ml PDGF α-receptor analog(dimeric or tetrameric) diluted in ELISA buffer B was added to each welland the plates were incubated over night at 4° C. Unbound protein wasremoved from the wells with two washes with ELISA buffer B. ¹²⁵I-labeledPDGF-AA was serially diluted in binding media (Hams F-12, 25 mM HEPES pH7.2, 0.25% rabbit serum albumin), and 100 μl of each dilution was addedto the wells. The samples were incubated for two hours at roomtemperature. Unbound ¹²⁵I-labeled PDGF-AA was removed with three washeswith binding media. One hundred microliters of 0.1 M citrate, pH 2.5 wasadded to each well, and the plates were incubated for five minutes.After the incubation, the citrate buffer was removed and transferred toa tube for counting in a gamma counter. Nonspecific binding for eachconcentration of ¹²⁵I-labeled PDGF-AA was determined by a parallel assaywherein separate wells coated only with goat anti-human IgG wereincubated with the ¹²⁵I-labeled PDGF-AA samples.

A saturation binding assay was performed on alpha T-7 cells transfectedwith the PDGF α-receptor. The cells were grown to confluency in 24-wellculture plates. The cells were washed one time with binding media.Iodinated PDGF-AA was serially diluted in binding media. One milliliterof each dilution was added to the wells, and the plates were incubatedfor 3 hours at 4° C. Unbound ¹²⁵I-labeled PDGF-AA was removed and thecells were washed three times with binding media. PBS containing 1%Triton X-100 was added to the cells for 5 minutes. The extracts wereharvested and counted in a gamma counter. Nonspecific binding wasdetermined at a single concentration of ¹²⁵I-labeled PDGF-AA using a500-fold excess PDGF-BB.

The dissociation constants determined by Scatchard analysis (ibid.) ofthe saturation binding assays for the tetrameric PDGF α-receptor analog,dimeric PDGF α-receptor analog and the control cells (Table 5).

TABLE 5 Dissociation Constants for the Tetrameric PDGF α-Receptor, theDimeric PDGF α-receptor and control cells Transfected with the PDGFα-receptor Receptor kD Tetrameric PDGF α-receptor analog  1.6 × 10⁻¹¹Dimeric PDGF α-receptor analog 8.51 × 10⁻¹¹ Control cells[PDGFα-receptor]  3.7 × 10⁻¹¹

A solid-phase competition binding assay was established using thetetrameric PDGF α-receptor analog. Ninety six-well microtiter plateswere coated with goat anti-human IgG (2 μg/ml), the wells blocked withELISA B buffer, 50 ng/ml of purified tetrameric PDGF α-receptor analogdiluted in binding media was; added, and the plates were incubated twohours at room temperature. Unbound receptor was removed and the wellswere washed with binding media. The plates were incubated for two hoursat room temperature with increasing concentrations of either PDGF-AA orPDGF-BB diluted in binding media. The wells were washed, then incubatedfor two hours at room temperature with 3 ng/ml ¹²⁵I-labeled PDGF-AAdiluted in binding media. Unbound labeled PDGF-AA was removed, the wellswere subsequently washed with binding media, and the bound, labeledPDGF-AA was harvested by the addition of 0.1 M citrate, pH 2.5, asdescribed for the saturation binding studies. PDGF-AB, PDGF-AA andPDGF-BB were found to compete for receptor binding with ¹²⁵I-PDGF-AA.

B. Use of Tetrameric PDGF α-Receptor Analogs As Antagonists forPDGF-Stimulated Mitogenesis

A tetrameric PDGF α-receptor analog, purified as described in Example16.B., was analyzed for the ability to neutralize PDGF-stimulatedmitogenesis in mouse 3T3 cells. Increasing amounts of the purifiedtetrameric PDGF α-receptor analog were mixed with PDGF-AA, -AB or -BBranging 0.6 to 5 ng. The mixtures were then added to cultures ofconfluent mouse 3T3 cells. The ability of the PDGF to stimulate amitogenic response, as measured by the incorporation of ³H-thymidine,was determined essentially as described (Raines and Ross, Methods inEnzymology 109: 749-773, 1985, which is incorporated by referenceherein). The tetrameric PDGF α-receptor analog demonstrated a doseresponse inhibition of PDGF-stimulated ³H-thymidine incorporation forall three isoforms of PDGF.

C. Inverse Ligand-Receptor Radioreceptor Assay

An inverse ligand-receptor radioreceptor assay was designed to screenfor the presence of PDGF-BB, PDGF-BB binding proteins, PDGF-BB relatedmolecules, and PDGF-β receptor antagonists in test samples. PDGF-BB (100ng/ml) was coated onto the walls of 96-well microtiter plates, and theplates were incubated at 4° C. for 16 hours. The wells were washed oncewith ELISA C buffer and then incubated with ELISA B buffer to block thenonspecific binding sites. To the wells were added 50 μl of either PDGFstandard or a test sample and 50 μl of ¹²⁵I-labeled tetrameric PDGFβ-receptor analog. The samples were incubated for one hour at roomtemperature. The wells were washed once with ELISA C buffer, and 0.1 Mcitrate, pH 2.5 containing 1% NP-40 was added to each well to disruptthe ligand-receptor analog bond and elute the bound receptor analog. Theacid wash was collected and counted in a gamma counter. The presence ofPDGF or a molecule which mimics PDGF or otherwise interferes with thebinding of the well-bound PDGF-BB with its receptor will cause adecrease in the binding of the radiolabeled tetrameric PDGF β-receptor.Using this assay, PDGF-BB was found to inhibit receptor binding whilePDGF-AA and PDGF-AB caused no significant decrease in receptor binding.

Example 18 Assay Methods

A. Preparation of Nitrocellulose Filters for Colony Assay

Colonies of transformants were tested for secretion of PDGF β-receptoranalogs by first growing the cells on nitrocellulose filters that hadbeen laid on top of solid growth medium. Nitrocellulose filters(Schleicher & Schuell, Keene, N.H.) were placed on top of solid growthmedium and were allowed to be completely wetted. Test colonies werepatched onto the wetted filters and were grown at 30° C. forapproximately 40 hours. The filters were then removed from the solidmedium, and the cells were removed by four successive rinses withWestern Transfer Buffer (Table 4). The nitrocellulose filters weresoaked in Western Buffer A (Table 4) for one hour at room temperature ona shaking platform with two changes of buffer. Secreted PDGFβ-R analogswere visualized on the filters described below.

B. Preparation of Protein Blot Filters

A nitrocellulose filter was soaked in Western Buffer A (Table 4) withoutthe gelatin and placed in a Minifold (Schleicher & Schuell, Keene,N.H.). Five milliliters of culture supernatant was added withoutdilution to the Minifold wells, and the liquid was allowed to passthrough the nitrocellulose filter by gravity. The nitrocellulose filterwas removed from the minifold and was soaked in Western Buffer A (Table3) for one hour on a shaking platform at room temperature. The bufferwas changed three times during the hour incubation.

C. Preparation of Western Blot Filters

The transformants were analyzed by Western blot, essentially asdescribed by Towbin et al. (Proc. Natl. Acad. Sci. USA 76: 4350-4354,1979) and Gordon et al. (U.S. Pat. No. 4,452,901). Culture supernatantsfrom appropriately grown transformants were diluted with three volumesof 95% ethanol. The ethanol mixtures were incubated overnight at −70° C.The precipitates were spun out of solution by centrifugation in an SS-24rotor at 18,000 rpm for 20 minutes. The supernatants were discarded andthe precipitate pellets were resuspended in 200 μl of dH₂O. Two hundredmicroliters of 2×loading buffer (Table 4) was added to each sample, andthe samples were incubated in a boiling water bath for 5 minutes.

The samples were electrophoresed in a 15% sodium dodecylsulfatepolyacrylamide gel under non-reducing conditions. The proteins wereelectrophoretically transferred to nitrocellulose paper using conditionsdescribed by Towbin et al. (ibid.). The nitrocellulose filters were thenincubated in Western Buffer A (Table 4) for 75 minutes at roomtemperature on a platform rocker.

D. Processing the Filters for Visualization with Antibody

Filters prepared as described above were screened for proteinsrecognized by the binding of a PDGF β-receptor specific monoclonalantibody, designated PR7212. The filters were removed from the WesternBuffer A (Table 4) and placed in sealed plastic bags containing a 10 mlsolution comprising 10 μg/ml PR7212 monoclonal antibody diluted inWestern Buffer A. The filters were incubated on a rocking platformovernight at 4° C. or for one hour at room temperature. Excess antibodywas removed with three 15-minute washes with Western Buffer A on ashaking platform at room temperature.

Ten microliters biotin-conjugated horse anti-mouse antibody (VectorLaboratories, Burlingame, Calif.) in 20 ml Western Buffer A was added tothe filters. The filters were re-incubated for one hour at roomtemperature on a platform shaker, and unbound conjugated antibody wasremoved with three fifteen-minute washes with Western Buffer A.

The filters were pre-incubated for one hour at room temperature with asolution comprising 50 μl Vectastain Reagent A (Vector Laboratories) in10 ml of Western Buffer A that had been allowed to incubate at roomtemperature for 30 minutes before use. The filters were washed with onequick wash with distilled water followed by three 15-minute washes withWestern Buffer B (Table 4) at room temperature. The Western Buffer Bwashes were followed by one wash with distilled water.

During the preceding wash step, the substrate reagent was prepared.Sixty mg of horseradish peroxidase reagent (Bio-Rad, Richmond, Calif.)was dissolved in 20 ml HPLC grade methanol. Ninety milliliters ofdistilled water was added to the dissolved peroxidase followed by 2.5 ml2 M Tris, pH 7.4 and 3.8 ml 4 M NaCl. One hundred microliters of 30%H₂O₂ was added just before use. The washed filters were incubated with75 ml of substrate and incubated at room temperature for 10 minutes withvigorous shaking. After the 10 minute incubation, the buffer waschanged, and the filters were incubated for an additional 10 minutes.The filters were then washed in distilled water for one hour at roomtemperature. Positives were scored as those samples which exhibitedcoloration.

E. Processing the Filters For Visualization with an Anti-Substance PAntibody

Filters prepared as described above were probed with an anti-substance Pantibody. The filters were removed from the Western Buffer A and rinsedwith Western transfer buffer, followed by a 5-minute wash in phosphatebuffered saline (PBS, Sigma, St. Louis, Mo.). The filters were incubatedwith a 10 ml solution containing 0.5 M 1-ethyl-3-3-dimethylamino propylcarbodiimide (Sigma) in 1.0 M NH₄Cl for 40 minutes at room temperature.After incubation, the filters were washed three times, for 5 minutes perwash, in PBS. The filters were blocked with 2% powdered milk diluted inPBS.

The filters were then incubated with a rat anti-substance P monoclonalantibody (Accurate Chemical & Scientific Corp., Westbury, N.Y.). Tenmicroliters of the antibody was diluted in 10 ml of antibody solution(PBS containing 20% fetal calf serum and 0.5% Tween-20). The filterswere incubated at room temperature for 1 hour. Unbound antibody wasremoved with four 5-minute washes with PBS.

The filters were then incubated with a biotin-conjugated rabbit anti-ratperoxidase antibody (Cappel Laboratories, Melvern, Pa.). The conjugatedantibody was diluted 1:1000 in 10 ml of antibody solution for 2 hours atroom temperature. Excess conjugated antibody was removed with four5-minute washes with PBS.

The filters were pre-incubated for 30 minutes at room temperature with asolution containing 50 μl Vectastain Reagent A (Vector Laboratories) and50 μl Vectastain Reagent B (Vector Laboratories) in 10 ml of antibodysolution that had been allowed to incubate for 30 minutes before use.Excess Vectastain reagents were removed by four 5-minute washes withPBS.

During the preceding wash step, the substrate reagent was prepared.Sixty milligrams of horseradish peroxidase reagent (Bio-RadLaboratories, Richmond, Calif.) was dissolved in 25 ml of HPLC grademethanol. Approximately 100 ml of PBS and 200 μl H₂O₂ were added justbefore use. The filters were incubated with the substrate reagent for 10to 20 minutes. The substrate was removed by a vigorous washing distilledwater.

F. Iodination of PDGF-BB

A PDGF-BB mutant molecule having a tyrosine replacing the phenylalanineat position 23 (PDGF-BB_(Tyr)) was iodinated and subsequently purified,using a purification method which produces 125I-labeled PDGF-BB with ahigher specific activity than primary-labeled material and which wasfound to substantially decrease the nonspecific binding component. ThePDGF-BB_(Tyr) was labeled using the Iodobead method (Pierce Chemical).The labeled protein was gel filtered over a C-25 desalting column(Pharmacia LKB Technologies) equilibrated with 10 mM acetic acid, 0.25%gelatin and 100 mM NaCl. The peak fractions were pooled and pH adjustedto 7.2 by the addition of Tris-base. The labeled mixture waschromatographed over an affinity column composed of PDGF β-receptoranalog protein coupled to CnBr-activated Sepharose (Pharmacia LKBTechnologies, Inc.). The column was washed with phosphate bufferedsaline and eluted with 0.1 M citrate, pH 2.5 containing 0.25% gelatin.The peak eluate fractions were pooled and assayed by ELISA to determinethe PDGF-BB concentration.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be evident that certain changes and modificationsmay be practiced within the scope of the appended claims.

36 4465 base pairs nucleic acid double linear cDNA N N Homo sapiensAdult Skin fibroblasts pR-rX1 CDS 354..3671 1 CCCTCAGCCC TGCTGCCCAGCACGAGCCTG TGCTCGCCCT GCCCAACGCA GACAGCCAGA 60 CCCAGGGCGG CCCCTCTGGCGGCTCTGCTC CTCCCGAAGG ATGCTTGGGG AGTGAGGCGA 120 AGCTGGGCGC TCCTCTCCCCTACAGCAGCC CCCTTCCTCC ATCCCTCTGT TCTCCTGAGC 180 CTTCAGGAGC CTGCACCAGTCCTGCCTGTC CTTCTACTCA GCTGTTACCC ACTCTGGGAC 240 CAGCAGTCTT TCTGATAACTGGGAGAGGGC AGTAAGGAGG ACTTCCTGGA GGGGGTGACT 300 GTCCAGAGCC TGGAACTGTGCCCACACCAG AAGCCATCAG CAGCAAGGAC ACC ATG 356 Met 1 CGG CTT CCG GGT GCGATG CCA GCT CTG GCC CTC AAA GGC GAG CTG CTG 404 Arg Leu Pro Gly Ala MetPro Ala Leu Ala Leu Lys Gly Glu Leu Leu 5 10 15 TTG CTG TCT CTC CTG TTACTT CTG GAA CCA CAG ATC TCT CAG GGC CTG 452 Leu Leu Ser Leu Leu Leu LeuLeu Glu Pro Gln Ile Ser Gln Gly Leu 20 25 30 GTC GTC ACA CCC CCG GGG CCAGAG CTT GTC CTC AAT GTC TCC AGC ACC 500 Val Val Thr Pro Pro Gly Pro GluLeu Val Leu Asn Val Ser Ser Thr 35 40 45 TTC GTT CTG ACC TGC TCG GGT TCAGCT CCG GTG GTG TGG GAA CGG ATG 548 Phe Val Leu Thr Cys Ser Gly Ser AlaPro Val Val Trp Glu Arg Met 50 55 60 65 TCC CAG GAG CCC CCA CAG GAA ATGGCC AAG GCC CAG GAT GGC ACC TTC 596 Ser Gln Glu Pro Pro Gln Glu Met AlaLys Ala Gln Asp Gly Thr Phe 70 75 80 TCC AGC GTG CTC ACA CTG ACC AAC CTCACT GGG CTA GAC ACG GGA GAA 644 Ser Ser Val Leu Thr Leu Thr Asn Leu ThrGly Leu Asp Thr Gly Glu 85 90 95 TAC TTT TGC ACC CAC AAT GAC TCC CGT GGACTG GAG ACC GAT GAG CGG 692 Tyr Phe Cys Thr His Asn Asp Ser Arg Gly LeuGlu Thr Asp Glu Arg 100 105 110 AAA CGG CTC TAC ATC TTT GTG CCA GAT CCCACC GTG GGC TTC CTC CCT 740 Lys Arg Leu Tyr Ile Phe Val Pro Asp Pro ThrVal Gly Phe Leu Pro 115 120 125 AAT GAT GCC GAG GAA CTA TTC ATC TTT CTCACG GAA ATA ACT GAG ATC 788 Asn Asp Ala Glu Glu Leu Phe Ile Phe Leu ThrGlu Ile Thr Glu Ile 130 135 140 145 ACC ATT CCA TGC CGA GTA ACA GAC CCACAG CTG GTG GTG ACA CTG CAC 836 Thr Ile Pro Cys Arg Val Thr Asp Pro GlnLeu Val Val Thr Leu His 150 155 160 GAG AAG AAA GGG GAC GTT GCA CTG CCTGTC CCC TAT GAT CAC CAA CGT 884 Glu Lys Lys Gly Asp Val Ala Leu Pro ValPro Tyr Asp His Gln Arg 165 170 175 GGC TTT TCT GGT ATC TTT GAG GAC AGAAGC TAC ATC TGC AAA ACC ACC 932 Gly Phe Ser Gly Ile Phe Glu Asp Arg SerTyr Ile Cys Lys Thr Thr 180 185 190 ATT GGG GAC AGG GAG GTG GAT TCT GATGCC TAC TAT GTC TAC AGA CTC 980 Ile Gly Asp Arg Glu Val Asp Ser Asp AlaTyr Tyr Val Tyr Arg Leu 195 200 205 CAG GTG TCA TCC ATC AAC GTC TCT GTGAAC GCA GTG CAG ACT GTG GTC 1028 Gln Val Ser Ser Ile Asn Val Ser Val AsnAla Val Gln Thr Val Val 210 215 220 225 CGC CAG GGT GAG AAC ATC ACC CTCATG TGC ATT GTG ATC GGG AAT GAG 1076 Arg Gln Gly Glu Asn Ile Thr Leu MetCys Ile Val Ile Gly Asn Glu 230 235 240 GTG GTC AAC TTC GAG TGG ACA TACCCC CGC AAA GAA AGT GGG CGG CTG 1124 Val Val Asn Phe Glu Trp Thr Tyr ProArg Lys Glu Ser Gly Arg Leu 245 250 255 GTG GAG CCG GTG ACT GAC TTC CTCTTG GAT ATG CCT TAC CAC ATC CGC 1172 Val Glu Pro Val Thr Asp Phe Leu LeuAsp Met Pro Tyr His Ile Arg 260 265 270 TCC ATC CTG CAC ATC CCC AGT GCCGAG TTA GAA GAC TCG GGG ACC TAC 1220 Ser Ile Leu His Ile Pro Ser Ala GluLeu Glu Asp Ser Gly Thr Tyr 275 280 285 ACC TGC AAT GTG ACG GAG AGT GTGAAT GAC CAT CAG GAT GAA AAG GCC 1268 Thr Cys Asn Val Thr Glu Ser Val AsnAsp His Gln Asp Glu Lys Ala 290 295 300 305 ATC AAC ATC ACC GTG GTT GAGAGC GGC TAC GTG CGG CTC CTG GGA GAG 1316 Ile Asn Ile Thr Val Val Glu SerGly Tyr Val Arg Leu Leu Gly Glu 310 315 320 GTG GGC ACA CTA CAA TTT GCTGAG CTG CAT CGG AGC CGG ACA CTG CAG 1364 Val Gly Thr Leu Gln Phe Ala GluLeu His Arg Ser Arg Thr Leu Gln 325 330 335 GTA GTG TTC GAG GCC TAC CCACCG CCC ACT GTC CTG TGG TTC AAA GAC 1412 Val Val Phe Glu Ala Tyr Pro ProPro Thr Val Leu Trp Phe Lys Asp 340 345 350 AAC CGC ACC CTG GGC GAC TCCAGC GCT GGC GAA ATC GCC CTG TCC ACG 1460 Asn Arg Thr Leu Gly Asp Ser SerAla Gly Glu Ile Ala Leu Ser Thr 355 360 365 CGC AAC GTG TCG GAG ACC CGGTAT GTG TCA GAG CTG ACA CTG GTT CGC 1508 Arg Asn Val Ser Glu Thr Arg TyrVal Ser Glu Leu Thr Leu Val Arg 370 375 380 385 GTG AAG GTG GCA GAG GCTGGC CAC TAC ACC ATG CGG GCC TTC CAT GAG 1556 Val Lys Val Ala Glu Ala GlyHis Tyr Thr Met Arg Ala Phe His Glu 390 395 400 GAT GCT GAG GTC CAG CTCTCC TTC CAG CTA CAG ATC AAT GTC CCT GTC 1604 Asp Ala Glu Val Gln Leu SerPhe Gln Leu Gln Ile Asn Val Pro Val 405 410 415 CGA GTG CTG GAG CTA AGTGAG AGC CAC CCT GAC AGT GGG GAA CAG ACA 1652 Arg Val Leu Glu Leu Ser GluSer His Pro Asp Ser Gly Glu Gln Thr 420 425 430 GTC CGC TGT CGT GGC CGGGGC ATG CCC CAG CCG AAC ATC ATC TGG TCT 1700 Val Arg Cys Arg Gly Arg GlyMet Pro Gln Pro Asn Ile Ile Trp Ser 435 440 445 GCC TGC AGA GAC CTC AAAAGG TGT CCA CGT GAG CTG CCG CCC ACG CTG 1748 Ala Cys Arg Asp Leu Lys ArgCys Pro Arg Glu Leu Pro Pro Thr Leu 450 455 460 465 CTG GGG AAC AGT TCCGAA GAG GAG AGC CAG CTG GAG ACT AAC GTG ACG 1796 Leu Gly Asn Ser Ser GluGlu Glu Ser Gln Leu Glu Thr Asn Val Thr 470 475 480 TAC TGG GAG GAG GAGCAG GAG TTT GAG GTG GTG AGC ACA CTG CGT CTG 1844 Tyr Trp Glu Glu Glu GlnGlu Phe Glu Val Val Ser Thr Leu Arg Leu 485 490 495 CAG CAC GTG GAT CGGCCA CTG TCG GTG CGC TGC ACG CTG CGC AAC GCT 1892 Gln His Val Asp Arg ProLeu Ser Val Arg Cys Thr Leu Arg Asn Ala 500 505 510 GTG GGC CAG GAC ACGCAG GAG GTC ATC GTG GTG CCA CAC TCC TTG CCC 1940 Val Gly Gln Asp Thr GlnGlu Val Ile Val Val Pro His Ser Leu Pro 515 520 525 TTT AAG GTG GTG GTGATC TCA GCC ATC CTG GCC CTG GTG GTG CTC ACC 1988 Phe Lys Val Val Val IleSer Ala Ile Leu Ala Leu Val Val Leu Thr 530 535 540 545 ATC ATC TCC CTTATC ATC CTC ATC ATG CTT TGG CAG AAG AAG CCA CGT 2036 Ile Ile Ser Leu IleIle Leu Ile Met Leu Trp Gln Lys Lys Pro Arg 550 555 560 TAC GAG ATC CGATGG AAG GTG ATT GAG TCT GTG AGC TCT GAC GGC CAT 2084 Tyr Glu Ile Arg TrpLys Val Ile Glu Ser Val Ser Ser Asp Gly His 565 570 575 GAG TAC ATC TACGTG GAC CCC ATG CAG CTG CCC TAT GAC TCC ACG TGG 2132 Glu Tyr Ile Tyr ValAsp Pro Met Gln Leu Pro Tyr Asp Ser Thr Trp 580 585 590 GAG CTG CCG CGGGAC CAG CTT GTG CTG GGA CGC ACC CTC GGC TCT GGG 2180 Glu Leu Pro Arg AspGln Leu Val Leu Gly Arg Thr Leu Gly Ser Gly 595 600 605 GCC TTT GGG CAGGTG GTG GAG GCC ACG GCT CAT GGC CTG AGC CAT TCT 2228 Ala Phe Gly Gln ValVal Glu Ala Thr Ala His Gly Leu Ser His Ser 610 615 620 625 CAG GCC ACGATG AAA GTG GCC GTC AAG ATG CTT AAA TCC ACA GCC CGC 2276 Gln Ala Thr MetLys Val Ala Val Lys Met Leu Lys Ser Thr Ala Arg 630 635 640 AGC AGT GAGAAG CAA GCC CTT ATG TCG GAG CTG AAG ATC ATG AGT CAC 2324 Ser Ser Glu LysGln Ala Leu Met Ser Glu Leu Lys Ile Met Ser His 645 650 655 CTT GGG CCCCAC CTG AAC GTG GTC AAC CTG TTG GGG GCC TGC ACC AAA 2372 Leu Gly Pro HisLeu Asn Val Val Asn Leu Leu Gly Ala Cys Thr Lys 660 665 670 GGA GGA CCCATC TAT ATC ATC ACT GAG TAC TGC CGC TAC GGA GAC CTG 2420 Gly Gly Pro IleTyr Ile Ile Thr Glu Tyr Cys Arg Tyr Gly Asp Leu 675 680 685 GTG GAC TACCTG CAC CGC AAC AAA CAC ACC TTC CTG CAG CAC CAC TCC 2468 Val Asp Tyr LeuHis Arg Asn Lys His Thr Phe Leu Gln His His Ser 690 695 700 705 GAC AAGCGC CGC CCG CCC AGC GCG GAG CTC TAC AGC AAT GCT CTG CCC 2516 Asp Lys ArgArg Pro Pro Ser Ala Glu Leu Tyr Ser Asn Ala Leu Pro 710 715 720 GTT GGGCTC CCC CTG CCC AGC CAT GTG TCC TTG ACC GGG GAG AGC GAC 2564 Val Gly LeuPro Leu Pro Ser His Val Ser Leu Thr Gly Glu Ser Asp 725 730 735 GGT GGCTAC ATG GAC ATG AGC AAG GAC GAG TCG GTG GAC TAT GTG CCC 2612 Gly Gly TyrMet Asp Met Ser Lys Asp Glu Ser Val Asp Tyr Val Pro 740 745 750 ATG CTGGAC ATG AAA GGA GAC GTC AAA TAT GCA GAC ATC GAG TCC TCC 2660 Met Leu AspMet Lys Gly Asp Val Lys Tyr Ala Asp Ile Glu Ser Ser 755 760 765 AAC TACATG GCC CCT TAC GAT AAC TAC GTT CCC TCT GCC CCT GAG AGG 2708 Asn Tyr MetAla Pro Tyr Asp Asn Tyr Val Pro Ser Ala Pro Glu Arg 770 775 780 785 ACCTGC CGA GCA ACT TTG ATC AAC GAG TCT CCA GTG CTA AGC TAC ATG 2756 Thr CysArg Ala Thr Leu Ile Asn Glu Ser Pro Val Leu Ser Tyr Met 790 795 800 GACCTC GTG GGC TTC AGC TAC CAG GTG GCC AAT GGC ATG GAG TTT CTG 2804 Asp LeuVal Gly Phe Ser Tyr Gln Val Ala Asn Gly Met Glu Phe Leu 805 810 815 GCCTCC AAG AAC TGC GTC CAC AGA GAC CTG GCG GCT AGG AAC GTG CTC 2852 Ala SerLys Asn Cys Val His Arg Asp Leu Ala Ala Arg Asn Val Leu 820 825 830 ATCTGT GAA GGC AAG CTG GTC AAG ATC TGT GAC TTT GGC CTG GCT CGA 2900 Ile CysGlu Gly Lys Leu Val Lys Ile Cys Asp Phe Gly Leu Ala Arg 835 840 845 GACATC ATG CGG GAC TCG AAT TAC ATC TCC AAA GGC AGC ACC TTT TTG 2948 Asp IleMet Arg Asp Ser Asn Tyr Ile Ser Lys Gly Ser Thr Phe Leu 850 855 860 865CCT TTA AAG TGG ATG GCT CCG GAG AGC ATC TTC AAC AGC CTC TAC ACC 2996 ProLeu Lys Trp Met Ala Pro Glu Ser Ile Phe Asn Ser Leu Tyr Thr 870 875 880ACC CTG AGC GAC GTG TGG TCC TTC GGG ATC CTG CTC TGG GAG ATC TTC 3044 ThrLeu Ser Asp Val Trp Ser Phe Gly Ile Leu Leu Trp Glu Ile Phe 885 890 895ACC TTG GGT GGC ACC CCT TAC CCA GAG CTG CCC ATG AAC GAG CAG TTC 3092 ThrLeu Gly Gly Thr Pro Tyr Pro Glu Leu Pro Met Asn Glu Gln Phe 900 905 910TAC AAT GCC ATC AAA CGG GGT TAC CGC ATG GCC CAG CCT GCC CAT GCC 3140 TyrAsn Ala Ile Lys Arg Gly Tyr Arg Met Ala Gln Pro Ala His Ala 915 920 925TCC GAC GAG ATC TAT GAG ATC ATG CAG AAG TGC TGG GAA GAG AAG TTT 3188 SerAsp Glu Ile Tyr Glu Ile Met Gln Lys Cys Trp Glu Glu Lys Phe 930 935 940945 GAG ATT CGG CCC CCC TTC TCC CAG CTG GTG CTG CTT CTC GAG AGA CTG 3236Glu Ile Arg Pro Pro Phe Ser Gln Leu Val Leu Leu Leu Glu Arg Leu 950 955960 TTG GGC GAA GGT TAC AAA AAG AAG TAC CAG CAG GTG GAT GAG GAG TTT 3284Leu Gly Glu Gly Tyr Lys Lys Lys Tyr Gln Gln Val Asp Glu Glu Phe 965 970975 CTG AGG AGT GAC CAC CCA GCC ATC CTT CGG TCC CAG GCC CGC TTG CCT 3332Leu Arg Ser Asp His Pro Ala Ile Leu Arg Ser Gln Ala Arg Leu Pro 980 985990 GGG TTC CAT GGC CTC CGA TCT CCC CTG GAC ACC AGC TCC GTC CTC TAT 3380Gly Phe His Gly Leu Arg Ser Pro Leu Asp Thr Ser Ser Val Leu Tyr 995 10001005 ACT GCC GTG CAG CCC AAT GAG GGT GAC AAC GAC TAT ATC ATC CCC CTG3428 Thr Ala Val Gln Pro Asn Glu Gly Asp Asn Asp Tyr Ile Ile Pro Leu1010 1015 1020 1025 CCT GAC CCC AAA CCC GAG GTT GCT GAC GAG GGC CCA CTGGAG GGT TCC 3476 Pro Asp Pro Lys Pro Glu Val Ala Asp Glu Gly Pro Leu GluGly Ser 1030 1035 1040 CCC AGC CTA GCC AGC TCC ACC CTG AAT GAA GTC AACACC TCC TCA ACC 3524 Pro Ser Leu Ala Ser Ser Thr Leu Asn Glu Val Asn ThrSer Ser Thr 1045 1050 1055 ATC TCC TGT GAC AGC CCC CTG GAG CCC CAG GACGAA CCA GAG CCA GAG 3572 Ile Ser Cys Asp Ser Pro Leu Glu Pro Gln Asp GluPro Glu Pro Glu 1060 1065 1070 CCC CAG CTT GAG CTC CAG GTG GAG CCG GAGCCA GAG CTG GAA CAG TTG 3620 Pro Gln Leu Glu Leu Gln Val Glu Pro Glu ProGlu Leu Glu Gln Leu 1075 1080 1085 CCG GAT TCG GGG TGC CCT GCG CCT CGGGCG GAA GCA GAG GAT AGC TTC 3668 Pro Asp Ser Gly Cys Pro Ala Pro Arg AlaGlu Ala Glu Asp Ser Phe 1090 1095 1100 1105 CTG TAGGGGGCTG GCCCCTACCCTGCCCTGCCT GAAGCTCCCC CCCTGCCAGC 3721 Leu ACCCAGCATC TCCTGGCCTGGCCTGACCGG GCTTCCTGTC AGCCAGGCTG CCCTTATCAG 3781 CTGTCCCCTT CTGGAAGCTTTCTGCTCCTG ACGTGTTGTG CCCCAAACCC TGGGGCTGGC 3841 TTAGGAGGCA AGAAAACTGCAGGGGCCGTG ACCAGCCCTC TGCCTCCAGG GAGGCCAACT 3901 GACTCTGAGC CAGGGTTCCCCCAGGGAACT CAGTTTTCCC ATATGTAAGA TGGGAAAGTT 3961 AGGCTTGATG ACCCAGAATCTAGGATTCTC TCCCTGGCTG ACACGGTGGG GAGACCGAAT 4021 CCCTCCCTGG GAAGATTCTTGGAGTTACTG AGGTGGTAAA TTAACATTTT TTCTGTTCAG 4081 CCAGCTACCC CTCAAGGAATCATAGCTCTC TCCTCGCACT TTTTATCCAC CCAGGAGCTA 4141 GGGAAGAGAC CCTAGCCTCCCTGGCTGCTG GCTGAGCTAG GGCCTAGCTT GAGCAGTGTT 4201 GCCTCATCCA GAAGAAAGCCAGTCTCCTCC CTATGATGCC AGTCCCTGCG TTCCCTGGCC 4261 CGAGCTGGTC TGGGGCCATTAGGCAGCCTA ATTAATGCTG GAGGCTGAGC CAAGTACAGG 4321 ACACCCCCAG CCTGCAGCCCTTGCCCAGGG CACTTGGAGC ACACGCAGCC ATAGCAAGTG 4381 CCTGTGTCCC TGTCCTTCAGGCCCATCAGT CCTGGGGCTT TTTCTTTATC ACCCTCAGTC 4441 TTAATCCATC CACCAGAGTCTAGA 4465 1106 amino acids amino acid linear protein not provided 2 MetArg Leu Pro Gly Ala Met Pro Ala Leu Ala Leu Lys Gly Glu Leu 1 5 10 15Leu Leu Leu Ser Leu Leu Leu Leu Leu Glu Pro Gln Ile Ser Gln Gly 20 25 30Leu Val Val Thr Pro Pro Gly Pro Glu Leu Val Leu Asn Val Ser Ser 35 40 45Thr Phe Val Leu Thr Cys Ser Gly Ser Ala Pro Val Val Trp Glu Arg 50 55 60Met Ser Gln Glu Pro Pro Gln Glu Met Ala Lys Ala Gln Asp Gly Thr 65 70 7580 Phe Ser Ser Val Leu Thr Leu Thr Asn Leu Thr Gly Leu Asp Thr Gly 85 9095 Glu Tyr Phe Cys Thr His Asn Asp Ser Arg Gly Leu Glu Thr Asp Glu 100105 110 Arg Lys Arg Leu Tyr Ile Phe Val Pro Asp Pro Thr Val Gly Phe Leu115 120 125 Pro Asn Asp Ala Glu Glu Leu Phe Ile Phe Leu Thr Glu Ile ThrGlu 130 135 140 Ile Thr Ile Pro Cys Arg Val Thr Asp Pro Gln Leu Val ValThr Leu 145 150 155 160 His Glu Lys Lys Gly Asp Val Ala Leu Pro Val ProTyr Asp His Gln 165 170 175 Arg Gly Phe Ser Gly Ile Phe Glu Asp Arg SerTyr Ile Cys Lys Thr 180 185 190 Thr Ile Gly Asp Arg Glu Val Asp Ser AspAla Tyr Tyr Val Tyr Arg 195 200 205 Leu Gln Val Ser Ser Ile Asn Val SerVal Asn Ala Val Gln Thr Val 210 215 220 Val Arg Gln Gly Glu Asn Ile ThrLeu Met Cys Ile Val Ile Gly Asn 225 230 235 240 Glu Val Val Asn Phe GluTrp Thr Tyr Pro Arg Lys Glu Ser Gly Arg 245 250 255 Leu Val Glu Pro ValThr Asp Phe Leu Leu Asp Met Pro Tyr His Ile 260 265 270 Arg Ser Ile LeuHis Ile Pro Ser Ala Glu Leu Glu Asp Ser Gly Thr 275 280 285 Tyr Thr CysAsn Val Thr Glu Ser Val Asn Asp His Gln Asp Glu Lys 290 295 300 Ala IleAsn Ile Thr Val Val Glu Ser Gly Tyr Val Arg Leu Leu Gly 305 310 315 320Glu Val Gly Thr Leu Gln Phe Ala Glu Leu His Arg Ser Arg Thr Leu 325 330335 Gln Val Val Phe Glu Ala Tyr Pro Pro Pro Thr Val Leu Trp Phe Lys 340345 350 Asp Asn Arg Thr Leu Gly Asp Ser Ser Ala Gly Glu Ile Ala Leu Ser355 360 365 Thr Arg Asn Val Ser Glu Thr Arg Tyr Val Ser Glu Leu Thr LeuVal 370 375 380 Arg Val Lys Val Ala Glu Ala Gly His Tyr Thr Met Arg AlaPhe His 385 390 395 400 Glu Asp Ala Glu Val Gln Leu Ser Phe Gln Leu GlnIle Asn Val Pro 405 410 415 Val Arg Val Leu Glu Leu Ser Glu Ser His ProAsp Ser Gly Glu Gln 420 425 430 Thr Val Arg Cys Arg Gly Arg Gly Met ProGln Pro Asn Ile Ile Trp 435 440 445 Ser Ala Cys Arg Asp Leu Lys Arg CysPro Arg Glu Leu Pro Pro Thr 450 455 460 Leu Leu Gly Asn Ser Ser Glu GluGlu Ser Gln Leu Glu Thr Asn Val 465 470 475 480 Thr Tyr Trp Glu Glu GluGln Glu Phe Glu Val Val Ser Thr Leu Arg 485 490 495 Leu Gln His Val AspArg Pro Leu Ser Val Arg Cys Thr Leu Arg Asn 500 505 510 Ala Val Gly GlnAsp Thr Gln Glu Val Ile Val Val Pro His Ser Leu 515 520 525 Pro Phe LysVal Val Val Ile Ser Ala Ile Leu Ala Leu Val Val Leu 530 535 540 Thr IleIle Ser Leu Ile Ile Leu Ile Met Leu Trp Gln Lys Lys Pro 545 550 555 560Arg Tyr Glu Ile Arg Trp Lys Val Ile Glu Ser Val Ser Ser Asp Gly 565 570575 His Glu Tyr Ile Tyr Val Asp Pro Met Gln Leu Pro Tyr Asp Ser Thr 580585 590 Trp Glu Leu Pro Arg Asp Gln Leu Val Leu Gly Arg Thr Leu Gly Ser595 600 605 Gly Ala Phe Gly Gln Val Val Glu Ala Thr Ala His Gly Leu SerHis 610 615 620 Ser Gln Ala Thr Met Lys Val Ala Val Lys Met Leu Lys SerThr Ala 625 630 635 640 Arg Ser Ser Glu Lys Gln Ala Leu Met Ser Glu LeuLys Ile Met Ser 645 650 655 His Leu Gly Pro His Leu Asn Val Val Asn LeuLeu Gly Ala Cys Thr 660 665 670 Lys Gly Gly Pro Ile Tyr Ile Ile Thr GluTyr Cys Arg Tyr Gly Asp 675 680 685 Leu Val Asp Tyr Leu His Arg Asn LysHis Thr Phe Leu Gln His His 690 695 700 Ser Asp Lys Arg Arg Pro Pro SerAla Glu Leu Tyr Ser Asn Ala Leu 705 710 715 720 Pro Val Gly Leu Pro LeuPro Ser His Val Ser Leu Thr Gly Glu Ser 725 730 735 Asp Gly Gly Tyr MetAsp Met Ser Lys Asp Glu Ser Val Asp Tyr Val 740 745 750 Pro Met Leu AspMet Lys Gly Asp Val Lys Tyr Ala Asp Ile Glu Ser 755 760 765 Ser Asn TyrMet Ala Pro Tyr Asp Asn Tyr Val Pro Ser Ala Pro Glu 770 775 780 Arg ThrCys Arg Ala Thr Leu Ile Asn Glu Ser Pro Val Leu Ser Tyr 785 790 795 800Met Asp Leu Val Gly Phe Ser Tyr Gln Val Ala Asn Gly Met Glu Phe 805 810815 Leu Ala Ser Lys Asn Cys Val His Arg Asp Leu Ala Ala Arg Asn Val 820825 830 Leu Ile Cys Glu Gly Lys Leu Val Lys Ile Cys Asp Phe Gly Leu Ala835 840 845 Arg Asp Ile Met Arg Asp Ser Asn Tyr Ile Ser Lys Gly Ser ThrPhe 850 855 860 Leu Pro Leu Lys Trp Met Ala Pro Glu Ser Ile Phe Asn SerLeu Tyr 865 870 875 880 Thr Thr Leu Ser Asp Val Trp Ser Phe Gly Ile LeuLeu Trp Glu Ile 885 890 895 Phe Thr Leu Gly Gly Thr Pro Tyr Pro Glu LeuPro Met Asn Glu Gln 900 905 910 Phe Tyr Asn Ala Ile Lys Arg Gly Tyr ArgMet Ala Gln Pro Ala His 915 920 925 Ala Ser Asp Glu Ile Tyr Glu Ile MetGln Lys Cys Trp Glu Glu Lys 930 935 940 Phe Glu Ile Arg Pro Pro Phe SerGln Leu Val Leu Leu Leu Glu Arg 945 950 955 960 Leu Leu Gly Glu Gly TyrLys Lys Lys Tyr Gln Gln Val Asp Glu Glu 965 970 975 Phe Leu Arg Ser AspHis Pro Ala Ile Leu Arg Ser Gln Ala Arg Leu 980 985 990 Pro Gly Phe HisGly Leu Arg Ser Pro Leu Asp Thr Ser Ser Val Leu 995 1000 1005 Tyr ThrAla Val Gln Pro Asn Glu Gly Asp Asn Asp Tyr Ile Ile Pro 1010 1015 1020Leu Pro Asp Pro Lys Pro Glu Val Ala Asp Glu Gly Pro Leu Glu Gly 10251030 1035 1040 Ser Pro Ser Leu Ala Ser Ser Thr Leu Asn Glu Val Asn ThrSer Ser 1045 1050 1055 Thr Ile Ser Cys Asp Ser Pro Leu Glu Pro Gln AspGlu Pro Glu Pro 1060 1065 1070 Glu Pro Gln Leu Glu Leu Gln Val Glu ProGlu Pro Glu Leu Glu Gln 1075 1080 1085 Leu Pro Asp Ser Gly Cys Pro AlaPro Arg Ala Glu Ala Glu Asp Ser 1090 1095 1100 Phe Leu 1105 57 basepairs nucleic acid single linear Other nucleic acid N N not providedZC871 3 ATTATACGCT CTCTTCCTCA GGTAAATGAG TGCCAGGGCC GGCAAGCCCC CGCTCCA57 56 base pairs nucleic acid single linear Other nucleic acid N N notprovided ZC872 4 CCGGGGAGCG GGGGCTTGCC GGCCCTGGCA CTCATTTACC TGAGGAAGAGAGAGCT 56 45 base pairs nucleic acid single linear Other nucleic acid NN not provided ZC904 5 CATGGGCACG TAATCTATAG ATTCATCCTT GCTCATATCC ATGTA45 38 base pairs nucleic acid single linear Other nucleic acid N N notprovided ZC906 6 AAGCTGTCCT CTGCTTCAGC CAGAGGTCCT GGGCAGCC 38 38 basepairs nucleic acid single linear Other nucleic acid N N not providedZC906 7 AAGCTGTCCT CTGCTTCAGC CAGAGGTCCT GGGCAGCC 38 21 base pairsnucleic acid single linear Other nucleic acid N N not provided ZC1380 8CATGGTGGAA TTCCTGCTGA T 21 29 base pairs nucleic acid single linearOther nucleic acid N N not provided ZC1447 9 TGGTTGTGCA GAGCTGAGGAAGAGATGGA 29 55 base pairs nucleic acid single linear Other nucleic acidN N not provided ZC1453 10 AATTCATTAT GTTGTTGCAA GCCTTCTTGT TCCTGCTAGCTGGTTTCGCT GTTAA 55 55 base pairs nucleic acid single linear Othernucleic acid N N not provided ZC1454 11 GATCTTAACA GCGAAACCAG CTAGCAGGAACAAGAAGGCT TGCAACAACA TAATG 55 21 base pairs nucleic acid single linearOther nucleic acid N N not provided ZC1478 12 ATCGCGAGCA TGCAGATCTG A 2125 base pairs nucleic acid single linear Other nucleic acid N N notprovided ZC1479 13 AGCTTCAGAT CTGCATGCTG CCGAT 25 52 base pairs nucleicacid single linear Other nucleic acid N N not provided ZC1776 14AGCTGAGCGC AAATGTTGTG TCGAGTGCCC ACCGTGCCCA GCTTAGAATT CT 52 52 basepairs nucleic acid single linear Other nucleic acid N N not providedZC1777 15 CTAGAGAATT CTAAGCTGGG CACGGTGGGC ACTCGACACA ACATTTGCGC TC 5295 base pairs nucleic acid single linear Other nucleic acid N N notprovided ZC1846 16 GATCGGCCAC TGTCGGTGCG CTGCACGCTG CGCAACGCTGTGGGCCAGGA CACGCAGGAG 60 GTCATCGTGG TGCCACACTC CTTGCCCTTT AAGCA 95 95base pairs nucleic acid single linear Other nucleic acid N N notprovided ZC1847 17 AGCTTGCTTA AAGGGCAAGG AGTGTGGCAC CACGATGACCTCCTGCGTGT CCTGGCCCAC 60 AGCGTTGCGC AGCGTGCAGC GCACCGACAG TGGCC 95 43base pairs nucleic acid single linear Other nucleic acid N N notprovided ZC1886 18 CCAGTGCCAA GCTTGTCTAG ACTTACCTTT AAAGGGCAAG GAG 43 11base pairs nucleic acid single linear Other nucleic acid N N notprovided ZC1892 19 AGCTTGAGCG T 11 11 base pairs nucleic acid singlelinear Other nucleic acid N N not provided ZC1893 20 CTAGACGCTC A 11 47base pairs nucleic acid single linear Other nucleic acid N N notprovided ZC1894 21 AGCTTCCAGT TCTTCGGCCT CATGTCAGTT CTTCGGCCTC ATGTGAT47 47 base pairs nucleic acid single linear Other nucleic acid N N notprovided ZC1895 22 CTAGATCACA TGAGGCCGAA GAACTGACAT GAGGCCGAAG AACTGGA47 66 base pairs nucleic acid single linear Other nucleic acid N N notprovided ZC2181 23 AATTCGGATC CACCATGGGC ACCAGCCACC CGGCGTTCCTGGTGTTAGGC TGCCTGCTGA 60 CCGGCC 66 71 base pairs nucleic acid singlelinear Other nucleic acid N N not provided ZC2182 24 TGAGCCTGATCCTGTGCCAA CTGAGCCTGC CATCGATCCT GCCAAACGAG AACGAGAAGG 60 TTGTGCAGCT A71 69 base pairs nucleic acid single linear Other nucleic acid N N notprovided ZC2183 25 AATTTAGCTG CACAACCTTC TCGTTCTCGT TTGGCAGGATCGATGGCAGG CTCAGTTGGC 60 ACAGGATCA 69 68 base pairs nucleic acid singlelinear Other nucleic acid N N not provided ZC2184 26 GGCTCAGGCCGGTCAGCAGG CAGCCTAACA CCAGGAACGC CGGGTGGCTG GTGCCCATGG 60 TGGATCCG 68 20base pairs nucleic acid single linear Other nucleic acid N N notprovided ZC2311 27 TGATCACCAT GGCTCAACTG 20 10 base pairs nucleic acidsingle linear Other nucleic acid N N not provided ZC2351 28 CGAATTCCAC10 26 base pairs nucleic acid single linear Other nucleic acid N N notprovided ZC2352 29 ATTATACGCA TGGTGGAATT CGAGCT 26 41 base pairs nucleicacid single linear Other nucleic acid N N not provided ZC2392 30ACGTAAGCTT GTCTAGACTT ACCTTCAGAA CGCAGGGTGG G 41 17 amino acids aminoacid linear peptide C-terminal not provided pWK1 31 Ala Leu His Asn HisTyr Thr Glu Lys Ser Leu Ser Leu Ser Pro Gly 1 5 10 15 Lys 22 base pairsnucleic acid single linear Other nucleic acid N N not provided 32TGTGACACTC TCCTGGGAGT TA 22 30 base pairs nucleic acid single linearOther nucleic acid N N not provided 33 GCATAGTAGT TACCATATCC TCTTGCACAG30 25 base pairs nucleic acid single linear Other nucleic acid N N notprovided 34 ACCGAACGTG AGAGGAGTGC TATAA 25 4054 base pairs nucleic aciddouble linear cDNA N N Homo sapiens p-alpha-17B CDS 205..3471 35GCCCTGGGGA CGGACCGTGG GCGGCGCGCA GCGGCGGGAC GCGTTTTGGG GACGTGGTGG 60CCAGCGCCTT CCTGCAGACC CACAGGGAAG TACTCCCTTT GACCTCCGGG GAGCTGCGAC 120CAGGTTATAC GTTGCTGGTG GAAAAGTGAC AATTCTAGGA AAAGAGCTAA AAGCCGGATC 180GGTGACCGAA AGTTTCCCAG AGCT ATG GGG ACT TCC CAT CCG GCG TTC CTG 231 MetGly Thr Ser His Pro Ala Phe Leu 1 5 GTC TTA GGC TGT CTT CTC ACA GGG CTGAGC CTA ATC CTC TGC CAG CTT 279 Val Leu Gly Cys Leu Leu Thr Gly Leu SerLeu Ile Leu Cys Gln Leu 10 15 20 25 TCA TTA CCC TCT ATC CTT CCA AAT GAAAAT GAA AAG GTT GTG CAG CTG 327 Ser Leu Pro Ser Ile Leu Pro Asn Glu AsnGlu Lys Val Val Gln Leu 30 35 40 AAT TCA TCC TTT TCT CTG AGA TGC TTT GGGGAG AGT GAA GTG AGC TGG 375 Asn Ser Ser Phe Ser Leu Arg Cys Phe Gly GluSer Glu Val Ser Trp 45 50 55 CAG TAC CCC ATG TCT GAA GAA GAG AGC TCC GATGTG GAA ATC AGA AAT 423 Gln Tyr Pro Met Ser Glu Glu Glu Ser Ser Asp ValGlu Ile Arg Asn 60 65 70 GAA GAA AAC AAC AGC GGC CTT TTT GTG ACG GTC TTGGAA GTG AGC AGT 471 Glu Glu Asn Asn Ser Gly Leu Phe Val Thr Val Leu GluVal Ser Ser 75 80 85 GCC TCG GCG GCC CAC ACA GGG TTG TAC ACT TGC TAT TACAAC CAC ACT 519 Ala Ser Ala Ala His Thr Gly Leu Tyr Thr Cys Tyr Tyr AsnHis Thr 90 95 100 105 CAG ACA GAA GAG AAT GAG CTT GAA GGC AGG CAC ATTTAC ATC TAT GTG 567 Gln Thr Glu Glu Asn Glu Leu Glu Gly Arg His Ile TyrIle Tyr Val 110 115 120 CCA GAC CCA GAT GTA GCC TTT GTA CCT CTA GGA ATGACG GAT TAT TTA 615 Pro Asp Pro Asp Val Ala Phe Val Pro Leu Gly Met ThrAsp Tyr Leu 125 130 135 GTC ATC GTG GAG GAT GAT GAT TCT GCC ATT ATA CCTTGT CGC ACA ACT 663 Val Ile Val Glu Asp Asp Asp Ser Ala Ile Ile Pro CysArg Thr Thr 140 145 150 GAT CCC GAG ACT CCT GTA ACC TTA CAC AAC AGT GAGGGG GTG GTA CCT 711 Asp Pro Glu Thr Pro Val Thr Leu His Asn Ser Glu GlyVal Val Pro 155 160 165 GCC TCC TAC GAC AGC AGA CAG GGC TTT AAT GGG ACCTTC ACT GTA GGG 759 Ala Ser Tyr Asp Ser Arg Gln Gly Phe Asn Gly Thr PheThr Val Gly 170 175 180 185 CCC TAT ATC TGT GAG GCC ACC GTC AAA GGA AAGAAG TTC CAG ACC ATC 807 Pro Tyr Ile Cys Glu Ala Thr Val Lys Gly Lys LysPhe Gln Thr Ile 190 195 200 CCA TTT AAT GTT TAT GCT TTA AAA GCA ACA TCAGAG CTG GAT CTA GAA 855 Pro Phe Asn Val Tyr Ala Leu Lys Ala Thr Ser GluLeu Asp Leu Glu 205 210 215 ATG GAA GCT CTT AAA ACC GTG TAT AAG TCA GGGGAA ACG ATT GTG GTC 903 Met Glu Ala Leu Lys Thr Val Tyr Lys Ser Gly GluThr Ile Val Val 220 225 230 ACC TGT GCT GTT TTT AAC AAT GAG GTG GTT GACCTT CAA TGG ACT TAC 951 Thr Cys Ala Val Phe Asn Asn Glu Val Val Asp LeuGln Trp Thr Tyr 235 240 245 CCT GGA GAA GTG AAA GGC AAA GGC ATC ACA ATACTG GAA GAA ATC AAA 999 Pro Gly Glu Val Lys Gly Lys Gly Ile Thr Ile LeuGlu Glu Ile Lys 250 255 260 265 GTC CCA TCC ATC AAA TTG GTG TAC ACT TTGACG GTC CCC GAG GCC ACG 1047 Val Pro Ser Ile Lys Leu Val Tyr Thr Leu ThrVal Pro Glu Ala Thr 270 275 280 GTG AAA GAC AGT GGA GAT TAC GAA TGT GCTGCC CGC CAG GCT ACC AGG 1095 Val Lys Asp Ser Gly Asp Tyr Glu Cys Ala AlaArg Gln Ala Thr Arg 285 290 295 GAG GTC AAA GAA ATG AAG AAA GTC ACT ATTTCT GTC CAT GAG AAA GGT 1143 Glu Val Lys Glu Met Lys Lys Val Thr Ile SerVal His Glu Lys Gly 300 305 310 TTC ATT GAA ATC AAA CCC ACC TTC AGC CAGTTG GAA GCT GTC AAC CTG 1191 Phe Ile Glu Ile Lys Pro Thr Phe Ser Gln LeuGlu Ala Val Asn Leu 315 320 325 CAT GAA GTC AAA CAT TTT GTT GTA GAG GTGCGG GCC TAC CCA CCT CCC 1239 His Glu Val Lys His Phe Val Val Glu Val ArgAla Tyr Pro Pro Pro 330 335 340 345 AGG ATA TCC TGG CTG AAA AAC AAT CTGACT CTG ATT GAA AAT CTC ACT 1287 Arg Ile Ser Trp Leu Lys Asn Asn Leu ThrLeu Ile Glu Asn Leu Thr 350 355 360 GAG ATC ACC ACT GAT GTG GAA AAG ATTCAG GAA ATA AGG TAT CGA AGC 1335 Glu Ile Thr Thr Asp Val Glu Lys Ile GlnGlu Ile Arg Tyr Arg Ser 365 370 375 AAA TTA AAG CTG ATC CGT GCT AAG GAAGAA GAC AGT GGC CAT TAT ACT 1383 Lys Leu Lys Leu Ile Arg Ala Lys Glu GluAsp Ser Gly His Tyr Thr 380 385 390 ATT GTA GCT CAA AAT GAA GAT GCT GTGAAG AGC TAT ACT TTT GAA CTG 1431 Ile Val Ala Gln Asn Glu Asp Ala Val LysSer Tyr Thr Phe Glu Leu 395 400 405 TTA ACT CAA GTT CCT TCA TCC ATT CTGGAC TTG GTC GAT GAT CAC CAT 1479 Leu Thr Gln Val Pro Ser Ser Ile Leu AspLeu Val Asp Asp His His 410 415 420 425 GGC TCA ACT GGG GGA CAG ACG GTGAGG TGC ACA GCT GAA GGC ACG CCG 1527 Gly Ser Thr Gly Gly Gln Thr Val ArgCys Thr Ala Glu Gly Thr Pro 430 435 440 CTT CCT GAT ATT GAG TGG ATG ATATGC AAA GAT ATT AAG AAA TGT AAT 1575 Leu Pro Asp Ile Glu Trp Met Ile CysLys Asp Ile Lys Lys Cys Asn 445 450 455 AAT GAA ACT TCC TGG ACT ATT TTGGCC AAC AAT GTC TCA AAC ATC ATC 1623 Asn Glu Thr Ser Trp Thr Ile Leu AlaAsn Asn Val Ser Asn Ile Ile 460 465 470 ACG GAG ATC CAC TCC CGA GAC AGGAGT ACC GTG GAG GGC CGT GTG ACT 1671 Thr Glu Ile His Ser Arg Asp Arg SerThr Val Glu Gly Arg Val Thr 475 480 485 TTC GCC AAA GTG GAG GAG ACC ATCGCC GTG CGA TGC CTG GCT AAG AAT 1719 Phe Ala Lys Val Glu Glu Thr Ile AlaVal Arg Cys Leu Ala Lys Asn 490 495 500 505 CTC CTT GGA GCT GAG AAC CGAGAG CTG AAG CTG GTG GCT CCC ACC CTG 1767 Leu Leu Gly Ala Glu Asn Arg GluLeu Lys Leu Val Ala Pro Thr Leu 510 515 520 CGT TCT GAA CTC ACG GTG GCTGCT GCA GTC CTG GTG CTG TTG GTG ATT 1815 Arg Ser Glu Leu Thr Val Ala AlaAla Val Leu Val Leu Leu Val Ile 525 530 535 GTG ATC ATC TCA CTT ATT GTCCTG GTT GTC ATT TGG AAA CAG AAA CCG 1863 Val Ile Ile Ser Leu Ile Val LeuVal Val Ile Trp Lys Gln Lys Pro 540 545 550 AGG TAT GAA ATT CGC TGG AGGGTC ATT GAA TCA ATC AGC CCG GAT GGA 1911 Arg Tyr Glu Ile Arg Trp Arg ValIle Glu Ser Ile Ser Pro Asp Gly 555 560 565 CAT GAA TAT ATT TAT GTG GACCCG ATG CAG CTG CCT TAT GAC TCA AGA 1959 His Glu Tyr Ile Tyr Val Asp ProMet Gln Leu Pro Tyr Asp Ser Arg 570 575 580 585 TGG GAG TTT CCA AGA GATGGA CTA GTG CTT GGT CGG GTC TTG GGG TCT 2007 Trp Glu Phe Pro Arg Asp GlyLeu Val Leu Gly Arg Val Leu Gly Ser 590 595 600 GGA GCG TTT GGG AAG GTGGTT GAA GGA ACA GCC TAT GGA TTA AGC CGG 2055 Gly Ala Phe Gly Lys Val ValGlu Gly Thr Ala Tyr Gly Leu Ser Arg 605 610 615 TCC CAA CCT GTC ATG AAAGTT GCA GTG AAG ATG CTA AAA CCC ACG GCC 2103 Ser Gln Pro Val Met Lys ValAla Val Lys Met Leu Lys Pro Thr Ala 620 625 630 AGA TCC AGT GAA AAA CAAGCT CTC ATG TCT GAA CTG AAG ATA ATG ACT 2151 Arg Ser Ser Glu Lys Gln AlaLeu Met Ser Glu Leu Lys Ile Met Thr 635 640 645 CAC CTG GGG CCA CAT TTGAAC ATT GTA AAC TTG CTG GGA GCC TGC ACC 2199 His Leu Gly Pro His Leu AsnIle Val Asn Leu Leu Gly Ala Cys Thr 650 655 660 665 AAG TCA GGC CCC ATTTAC ATC ATC ACA GAG TAT TGC TTC TAT GGA GAT 2247 Lys Ser Gly Pro Ile TyrIle Ile Thr Glu Tyr Cys Phe Tyr Gly Asp 670 675 680 TTG GTC AAC TAT TTGCAT AAG AAT AGG GAT AGC TTC CTG AGC CAC CAC 2295 Leu Val Asn Tyr Leu HisLys Asn Arg Asp Ser Phe Leu Ser His His 685 690 695 CCA GAG AAG CCA AAGAAA GAG CTG GAT ATC TTT GGA TTG AAC CCT GCT 2343 Pro Glu Lys Pro Lys LysGlu Leu Asp Ile Phe Gly Leu Asn Pro Ala 700 705 710 GAT GAA AGC ACA CGGAGC TAT GTT ATT TTA TCT TTT GAA AAC AAT GGT 2391 Asp Glu Ser Thr Arg SerTyr Val Ile Leu Ser Phe Glu Asn Asn Gly 715 720 725 GAC TAC ATG GAC ATGAAG CAG GCT GAT ACT ACA CAG TAT GTC CCC ATG 2439 Asp Tyr Met Asp Met LysGln Ala Asp Thr Thr Gln Tyr Val Pro Met 730 735 740 745 CTA GAA AGG AAAGAG GTT TCT AAA TAT TCC GAC ATC CAG AGA TCA CTC 2487 Leu Glu Arg Lys GluVal Ser Lys Tyr Ser Asp Ile Gln Arg Ser Leu 750 755 760 TAT GAT CGT CCAGCC TCA TAT AAG AAG AAA TCT ATG TTA GAC TCA GAA 2535 Tyr Asp Arg Pro AlaSer Tyr Lys Lys Lys Ser Met Leu Asp Ser Glu 765 770 775 GTC AAA AAC CTCCTT TCA GAT GAT AAC TCA GAA GGC CTT ACT TTA TTG 2583 Val Lys Asn Leu LeuSer Asp Asp Asn Ser Glu Gly Leu Thr Leu Leu 780 785 790 GAT TTG TTG AGCTTC ACC TAT CAA GTT GCC CGA GGA ATG GAG TTT TTG 2631 Asp Leu Leu Ser PheThr Tyr Gln Val Ala Arg Gly Met Glu Phe Leu 795 800 805 GCT TCA AAA AATTGT GTC CAC CGT GAT CTG GCT GCT CGC AAC GTC CTC 2679 Ala Ser Lys Asn CysVal His Arg Asp Leu Ala Ala Arg Asn Val Leu 810 815 820 825 CTG GCA CAAGGA AAA ATT GTG AAG ATC TGT GAC TTT GGC CTG GCC AGA 2727 Leu Ala Gln GlyLys Ile Val Lys Ile Cys Asp Phe Gly Leu Ala Arg 830 835 840 GAC ATC ATGCAT GAT TCG AAC TAT GTG TCG AAA GGC AGT ACC TTT CTG 2775 Asp Ile Met HisAsp Ser Asn Tyr Val Ser Lys Gly Ser Thr Phe Leu 845 850 855 CCC GTG AAGTGG ATG GCT CCT GAG AGC ATC TTT GAC AAC CTC TAC ACC 2823 Pro Val Lys TrpMet Ala Pro Glu Ser Ile Phe Asp Asn Leu Tyr Thr 860 865 870 ACA CTG AGTGAT GTC TGG TCT TAT GGC ATT CTG CTC TGG GAG ATC TTT 2871 Thr Leu Ser AspVal Trp Ser Tyr Gly Ile Leu Leu Trp Glu Ile Phe 875 880 885 TCC CTT GGTGGC ACC CCT TAC CCC GGC ATG ATG GTG GAT TCT ACT TTC 2919 Ser Leu Gly GlyThr Pro Tyr Pro Gly Met Met Val Asp Ser Thr Phe 890 895 900 905 TAC AATAAG ATC AAG AGT GGG TAC CGG ATG GCC AAG CCT GAC CAC GCT 2967 Tyr Asn LysIle Lys Ser Gly Tyr Arg Met Ala Lys Pro Asp His Ala 910 915 920 ACC AGTGAA GTC TAC GAG ATC ATG GTG AAA TGC TGG AAC AGT GAG CCG 3015 Thr Ser GluVal Tyr Glu Ile Met Val Lys Cys Trp Asn Ser Glu Pro 925 930 935 GAG AAGAGA CCC TCC TTT TAC CAC CTG AGT GAG ATT GTG GAG AAT CTG 3063 Glu Lys ArgPro Ser Phe Tyr His Leu Ser Glu Ile Val Glu Asn Leu 940 945 950 CTG CCTGGA CAA TAT AAA AAG AGT TAT GAA AAA ATT CAC CTG GAC TTC 3111 Leu Pro GlyGln Tyr Lys Lys Ser Tyr Glu Lys Ile His Leu Asp Phe 955 960 965 CTG AAGAGT GAC CAT CCT GCT GTG GCA CGC ATG CGT GTG GAC TCA GAC 3159 Leu Lys SerAsp His Pro Ala Val Ala Arg Met Arg Val Asp Ser Asp 970 975 980 985 AATGCA TAC ATT GGT GTC ACC TAC AAA AAC GAG GAA GAC AAG CTG AAG 3207 Asn AlaTyr Ile Gly Val Thr Tyr Lys Asn Glu Glu Asp Lys Leu Lys 990 995 1000 GACTGG GAG GGT GGT CTG GAT GAG CAG AGA CTG AGC GCT GAC AGT GGC 3255 Asp TrpGlu Gly Gly Leu Asp Glu Gln Arg Leu Ser Ala Asp Ser Gly 1005 1010 1015TAC ATC ATT CCT CTG CCT GAC ATT GAC CCT GTC CCT GAG GAG GAG GAC 3303 TyrIle Ile Pro Leu Pro Asp Ile Asp Pro Val Pro Glu Glu Glu Asp 1020 10251030 CTG GGC AAG AGG AAC AGA CAC AGC TCG CAG ACC TCT GAA GAG AGT GCC3351 Leu Gly Lys Arg Asn Arg His Ser Ser Gln Thr Ser Glu Glu Ser Ala1035 1040 1045 ATT GAG ACG GGT TCC AGC AGT TCC ACC TTC ATC AAG AGA GAGGAC GAG 3399 Ile Glu Thr Gly Ser Ser Ser Ser Thr Phe Ile Lys Arg Glu AspGlu 1050 1055 1060 1065 ACC ATT GAA GAC ATC GAC ATG ATG GAC GAC ATC GGCATA GAC TCT TCA 3447 Thr Ile Glu Asp Ile Asp Met Met Asp Asp Ile Gly IleAsp Ser Ser 1070 1075 1080 GAC CTG GTG GAA GAC AGC TTC CTG TAACTGGCGGATTCGAGGGG TTCCTTCCAC 3501 Asp Leu Val Glu Asp Ser Phe Leu 1085TTCTGGGGCC ACCTCTGGAT CCCGTTCAGA AAACCACTTT ATTGCAATGC GGAGGTTGAG 3561AGGAGGACTT GGTTGATGTT TAAAGAGAAG TTCCCAGCCA AGGGCCTCGG GGAGCGTTCT 3621AAATATGAAT GAATGGGATA TTTTGAAATG AACTTTGTCA GTGTTGCCTC TTGCAATGCC 3681TCAGTAGCAT CTCAGTGGTG TGTGAAGTTT GGAGATAGAT GGATAAGGGA ATAATAGGCC 3741ACAGAAGGTG AACTTTGTGC TTCAAGGACA TTGGTGAGAG TCCAACAGAC ACAATTTATA 3801CTGCGACAGA ACTTCAGCAT TGTAATTATG TAAATAACTC TAACCAAGGC TGTGTTTAGA 3861TTGTATTAAC TATCTTCTTT GGACTTCTGA AGAGACCACT CAATCCATCC TGTACTTCCC 3921TCTTGAAACC TGATGTAGCT GCTGTTGAAC TTTTTAAAGA AGTGCATGAA AAACCATTTT 3981TGAACCTTAA AAGGTACTGG TACTATAGCA TTTTGCTATC TTTTTTAGTG TTAAAGAGAT 4041AAAGAATAAT AAG 4054 1089 amino acids amino acid linear protein notprovided 36 Met Gly Thr Ser His Pro Ala Phe Leu Val Leu Gly Cys Leu LeuThr 1 5 10 15 Gly Leu Ser Leu Ile Leu Cys Gln Leu Ser Leu Pro Ser IleLeu Pro 20 25 30 Asn Glu Asn Glu Lys Val Val Gln Leu Asn Ser Ser Phe SerLeu Arg 35 40 45 Cys Phe Gly Glu Ser Glu Val Ser Trp Gln Tyr Pro Met SerGlu Glu 50 55 60 Glu Ser Ser Asp Val Glu Ile Arg Asn Glu Glu Asn Asn SerGly Leu 65 70 75 80 Phe Val Thr Val Leu Glu Val Ser Ser Ala Ser Ala AlaHis Thr Gly 85 90 95 Leu Tyr Thr Cys Tyr Tyr Asn His Thr Gln Thr Glu GluAsn Glu Leu 100 105 110 Glu Gly Arg His Ile Tyr Ile Tyr Val Pro Asp ProAsp Val Ala Phe 115 120 125 Val Pro Leu Gly Met Thr Asp Tyr Leu Val IleVal Glu Asp Asp Asp 130 135 140 Ser Ala Ile Ile Pro Cys Arg Thr Thr AspPro Glu Thr Pro Val Thr 145 150 155 160 Leu His Asn Ser Glu Gly Val ValPro Ala Ser Tyr Asp Ser Arg Gln 165 170 175 Gly Phe Asn Gly Thr Phe ThrVal Gly Pro Tyr Ile Cys Glu Ala Thr 180 185 190 Val Lys Gly Lys Lys PheGln Thr Ile Pro Phe Asn Val Tyr Ala Leu 195 200 205 Lys Ala Thr Ser GluLeu Asp Leu Glu Met Glu Ala Leu Lys Thr Val 210 215 220 Tyr Lys Ser GlyGlu Thr Ile Val Val Thr Cys Ala Val Phe Asn Asn 225 230 235 240 Glu ValVal Asp Leu Gln Trp Thr Tyr Pro Gly Glu Val Lys Gly Lys 245 250 255 GlyIle Thr Ile Leu Glu Glu Ile Lys Val Pro Ser Ile Lys Leu Val 260 265 270Tyr Thr Leu Thr Val Pro Glu Ala Thr Val Lys Asp Ser Gly Asp Tyr 275 280285 Glu Cys Ala Ala Arg Gln Ala Thr Arg Glu Val Lys Glu Met Lys Lys 290295 300 Val Thr Ile Ser Val His Glu Lys Gly Phe Ile Glu Ile Lys Pro Thr305 310 315 320 Phe Ser Gln Leu Glu Ala Val Asn Leu His Glu Val Lys HisPhe Val 325 330 335 Val Glu Val Arg Ala Tyr Pro Pro Pro Arg Ile Ser TrpLeu Lys Asn 340 345 350 Asn Leu Thr Leu Ile Glu Asn Leu Thr Glu Ile ThrThr Asp Val Glu 355 360 365 Lys Ile Gln Glu Ile Arg Tyr Arg Ser Lys LeuLys Leu Ile Arg Ala 370 375 380 Lys Glu Glu Asp Ser Gly His Tyr Thr IleVal Ala Gln Asn Glu Asp 385 390 395 400 Ala Val Lys Ser Tyr Thr Phe GluLeu Leu Thr Gln Val Pro Ser Ser 405 410 415 Ile Leu Asp Leu Val Asp AspHis His Gly Ser Thr Gly Gly Gln Thr 420 425 430 Val Arg Cys Thr Ala GluGly Thr Pro Leu Pro Asp Ile Glu Trp Met 435 440 445 Ile Cys Lys Asp IleLys Lys Cys Asn Asn Glu Thr Ser Trp Thr Ile 450 455 460 Leu Ala Asn AsnVal Ser Asn Ile Ile Thr Glu Ile His Ser Arg Asp 465 470 475 480 Arg SerThr Val Glu Gly Arg Val Thr Phe Ala Lys Val Glu Glu Thr 485 490 495 IleAla Val Arg Cys Leu Ala Lys Asn Leu Leu Gly Ala Glu Asn Arg 500 505 510Glu Leu Lys Leu Val Ala Pro Thr Leu Arg Ser Glu Leu Thr Val Ala 515 520525 Ala Ala Val Leu Val Leu Leu Val Ile Val Ile Ile Ser Leu Ile Val 530535 540 Leu Val Val Ile Trp Lys Gln Lys Pro Arg Tyr Glu Ile Arg Trp Arg545 550 555 560 Val Ile Glu Ser Ile Ser Pro Asp Gly His Glu Tyr Ile TyrVal Asp 565 570 575 Pro Met Gln Leu Pro Tyr Asp Ser Arg Trp Glu Phe ProArg Asp Gly 580 585 590 Leu Val Leu Gly Arg Val Leu Gly Ser Gly Ala PheGly Lys Val Val 595 600 605 Glu Gly Thr Ala Tyr Gly Leu Ser Arg Ser GlnPro Val Met Lys Val 610 615 620 Ala Val Lys Met Leu Lys Pro Thr Ala ArgSer Ser Glu Lys Gln Ala 625 630 635 640 Leu Met Ser Glu Leu Lys Ile MetThr His Leu Gly Pro His Leu Asn 645 650 655 Ile Val Asn Leu Leu Gly AlaCys Thr Lys Ser Gly Pro Ile Tyr Ile 660 665 670 Ile Thr Glu Tyr Cys PheTyr Gly Asp Leu Val Asn Tyr Leu His Lys 675 680 685 Asn Arg Asp Ser PheLeu Ser His His Pro Glu Lys Pro Lys Lys Glu 690 695 700 Leu Asp Ile PheGly Leu Asn Pro Ala Asp Glu Ser Thr Arg Ser Tyr 705 710 715 720 Val IleLeu Ser Phe Glu Asn Asn Gly Asp Tyr Met Asp Met Lys Gln 725 730 735 AlaAsp Thr Thr Gln Tyr Val Pro Met Leu Glu Arg Lys Glu Val Ser 740 745 750Lys Tyr Ser Asp Ile Gln Arg Ser Leu Tyr Asp Arg Pro Ala Ser Tyr 755 760765 Lys Lys Lys Ser Met Leu Asp Ser Glu Val Lys Asn Leu Leu Ser Asp 770775 780 Asp Asn Ser Glu Gly Leu Thr Leu Leu Asp Leu Leu Ser Phe Thr Tyr785 790 795 800 Gln Val Ala Arg Gly Met Glu Phe Leu Ala Ser Lys Asn CysVal His 805 810 815 Arg Asp Leu Ala Ala Arg Asn Val Leu Leu Ala Gln GlyLys Ile Val 820 825 830 Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp Ile MetHis Asp Ser Asn 835 840 845 Tyr Val Ser Lys Gly Ser Thr Phe Leu Pro ValLys Trp Met Ala Pro 850 855 860 Glu Ser Ile Phe Asp Asn Leu Tyr Thr ThrLeu Ser Asp Val Trp Ser 865 870 875 880 Tyr Gly Ile Leu Leu Trp Glu IlePhe Ser Leu Gly Gly Thr Pro Tyr 885 890 895 Pro Gly Met Met Val Asp SerThr Phe Tyr Asn Lys Ile Lys Ser Gly 900 905 910 Tyr Arg Met Ala Lys ProAsp His Ala Thr Ser Glu Val Tyr Glu Ile 915 920 925 Met Val Lys Cys TrpAsn Ser Glu Pro Glu Lys Arg Pro Ser Phe Tyr 930 935 940 His Leu Ser GluIle Val Glu Asn Leu Leu Pro Gly Gln Tyr Lys Lys 945 950 955 960 Ser TyrGlu Lys Ile His Leu Asp Phe Leu Lys Ser Asp His Pro Ala 965 970 975 ValAla Arg Met Arg Val Asp Ser Asp Asn Ala Tyr Ile Gly Val Thr 980 985 990Tyr Lys Asn Glu Glu Asp Lys Leu Lys Asp Trp Glu Gly Gly Leu Asp 995 10001005 Glu Gln Arg Leu Ser Ala Asp Ser Gly Tyr Ile Ile Pro Leu Pro Asp1010 1015 1020 Ile Asp Pro Val Pro Glu Glu Glu Asp Leu Gly Lys Arg AsnArg His 1025 1030 1035 1040 Ser Ser Gln Thr Ser Glu Glu Ser Ala Ile GluThr Gly Ser Ser Ser 1045 1050 1055 Ser Thr Phe Ile Lys Arg Glu Asp GluThr Ile Glu Asp Ile Asp Met 1060 1065 1070 Met Asp Asp Ile Gly Ile AspSer Ser Asp Leu Val Glu Asp Ser Phe 1075 1080 1085 Leu

What is claimed is:
 1. A method for producing a secreted ligand-bindingfusion protein comprising: (a) introducing into a host cell a DNAconstruct comprising a transcriptional promoter operatively linked to asecretory signal sequence followed downstream by and in proper readingframe with a DNA sequence encoding a polypeptide chain that comprises(i) a ligand-binding domain of a receptor, wherein the receptor is areceptor for a growth factor or a hormone, and (ii) an immunoglobulinconstant region polypeptide; and (b) culturing the host cell in anappropriate culture medium whereby the ligand-binding fusion protein issecreted, wherein the ligand-binding fusion protein binds the growthfactor or the hormone.
 2. The method of claim 1, further comprising: (c)obtaining the ligand-binding fusion protein from the culture medium ofthe cultured host cell.
 3. The method of claim 1, wherein the ligandbinding fusion protein competitively binds the growth factor or thehormone.
 4. The method of claim 1, wherein the immunoglobulin constantregion polypeptide comprises an immunoglobulin heavy chain constantregion domain.
 5. The method of claim 4, wherein the immunoglobulinheavy chain constant region domain is an IgG heavy chain constant regiondomain.
 6. The method of claim 4, wherein the polypeptide chain furthercomprises an immunoglobulin hinge region joined to the immunoglobulinheavy chain constant region domain.
 7. The method of claim 1, whereinthe ligand-binding fusion protein is a dimer.
 8. The method of claim 7,wherein the dimer is a homodimer.
 9. A method for producing a secretedligand-binding fusion protein comprising: (a) culturing a host cell inan appropriate culture medium, wherein the host cell comprises a DNAconstruct comprising a transcriptional promoter operatively linked to asecretory signal sequence followed downstream by and in proper readingframe with a DNA sequence encoding a polypeptide chain that comprises(i) a ligand-binding domain of a receptor, wherein the receptor, and(ii) an immunoglobulin constant region polypeptide, whereby theligand-binding fusion protein is secreted; and (b) obtaining theligand-binding fusion protein from the medium of the cultured host cell,wherein the ligand-binding fusion protein binds the growth factor or thehormone.